Cloned tetrodotoxin-sensitive sodium channel α-subunit and a splice variant thereof

ABSTRACT

DNA encoding for a voltage-gated, TTX-sensitive sodium channel is isolated. Also disclosed are polypeptide products of recombinant expression of these DNA sequences, expression vectors comprising the DNA sequences, and host cells transformed with these expression vectors. Other aspects of this invention are peptides whose sequences are based on the amino acid sequences deduced from these DNA sequences, antibodies specific for such proteins and peptides, procedures for detection and quantitation of such proteins, and nucleic acids related thereto. Another aspect of this invention is the use of this voltage-gated, tetrodotoxin-sensitive sodium channel as a therapeutic target for compounds.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 09/024,020, filed on Feb. 16, 1998, now U.S. Pat. No. 6,030,810, which in turn claims the benefit under 35 U.S.C. 119(e) of prior provisional U.S. patent application Ser. No. 60/039,447, filed on Feb. 26, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to sodium channel proteins and more particularly to a novel cloned α-subunit of a voltage-gated, tetrodotoxin-sensitive sodium channel protein. This invention further relates to the production of this protein by recombinant technology and nucleic acid sequences encoding for this protein.

2. Previous Art

The basic unit of information transmitted from one part of the nervous system to another is a single action potential or nerve impulse. The “transmission line” for these impulses is the axon, or nerve fiber. The electrical excitability of the nerve membrane has been shown to depend on the membrane's voltage-sensitive ionic permeability system that allows it to use energy stored in ionic concentration gradients. Electrical activity of the nerve is triggered by a depolarization of the membrane, which opens channels through the membrane that are highly selective for sodium ions, which are then driven inward by the electrochemical gradient. Of the many ionic channels, the voltage-gated or voltage-sensitive sodium channel is one of the most studied. It is a transmembrane protein that is essential for the generation of action potentials in excitable cells. An excellent review of sodium channels is presented in Catterall, TINS 16(12):500-506 (1993).

The cDNAs for several Na⁺ channels have been cloned and sequenced. Numa, et al., Annals of the New York Academy of Sciences 479:338-355 (1986), describe cDNA from the electric organ of eel and two different cDNAs from rat-brain. Rogart, U.S. Pat. No. 5,380,836, describes cDNA from rat cardiac tissue. See also Rogart, Cribbs, et al., Proc. Natl. Acad. Sci., 86:8170-8174 (1989). A peripheral nerve sodium channel, referred to as PN1, has been detected based on sodium current studies and hybridization to a highly conserved sodium channel probe by D'Arcangelo, et al., J. Cell Biol. 122:915-921 (1993), and subsequently cloned from PC12 cells, Toledo-Aral, et al., Proc. Nat. Acad. Sci. 94:1527-1532 (1997). The sequence of rat PN1 cloned from dorsal root ganglia and its functional expression have been described, Sangameswaran, et al., J. Biol. Chem 272:14805-14809 (1997). Other cloned sodium channels include rat brain types IIa, Auld, et al., Neuron 1:449-461 (1988), and III, Kayano, et al., FEBS Lett. 228:187-194 (1988), rat skeletal muscle, Trimmer, et al., Neuron 3:33-49 (1989), rat NaCh6, Schaller, et al., J. Neurosci. 15:3231-3242 (1995), rat peripheral nerve sodium channel type 3 (rPN3), Sangameswaran, et al., J. Biol Chem. 271:5953-5956 (1996), also called SNS, Akopian, et al., Nature 379:257-262 (1996), rat atypical channel, Felipe, et al., J. Biol. Chem. 269:30125-30131 (1994), and the rat glial sodium channel, Akopian, et al., FEBS Lett. 400:183-187 (1997).

These studies have shown that the amino acid sequence of the Na⁺ channel has been conserved over a long evolutionary period. These studies have also revealed that the channel is a single polypeptide containing four internal repeats, or homologous domains (domains I-IV), having similar amino acid sequences. Each domain folds into six predicted transmembrane α-helices or segments: five are hydrophobic segments and one segment is highly charged with many lysine and arginine residues. This highly charged segment is the fourth transmembrane segment in each domain (the S4 segment) and is likely to be involved in voltage-gating. The positively charged side chains on the S4 segment are likely to be paired with the negatively charged side chains on the other five segments, such that membrane depolarization could shift the position of one helix relative to the other, thereby opening the channel. Accessory subunits may modify the function of the channel.

Therapeutic utilities in recombinant materials derived from the DNA of the numerous sodium channels have been discovered. For example, Cherksey, U.S. Pat. No. 5,132,296, discloses purified Na⁺ channels that have proven useful as therapeutic and diagnostic tools.

Isoforms of sodium channels are divided into “subfamilies”. The term “isoform” is used to mean distinct but closely related sodium channel proteins, i.e., those having an amino acid homology of approximately 60-80%. These isoforms also show strong homology in functions. The term “subfamilies” is used to mean distinct sodium channels that have an amino acid homology of approximately 80-95%. Combinations of several factors are used to determine the distinctions within a subfamily, for example, the speed of a channel, chromosomal location, expression data, homology to other channels within a species, and homology to a channel of the same subfamily across species. Another consideration is an affinity to tetrodotoxin (“TTX”). TTX is a highly potent toxin from the puffer or fugu fish which blocks the conduction of nerve impulses along axons and in excitable membranes of nerve fibers. TTX binds to the Na⁺ channel and blocks the flow of sodium ions.

Studies using TTX as a probe have shed much light on the mechanism and structure of Na⁺ channels. There are three Na⁺ channel subtypes that are defined by the affinity for TTX, which can be measured by the IC₅₀ values: TTX-sensitive Na⁺ channels (IC₅₀≈1-30 nM), TTX-insensitive N⁺ channels (IC₅₀≈1-5 μm), and TTX-resistant Na⁺ channels (IC₅₀≧100 μM).

TTX-insensitive action potentials were first studied in rat skeletal muscle. See Redfern, et al., Acta Physiol. Scand. 82:70-78 (1971). Subsequently, these action potentials were described in other mammalian tissues, including newborn mammalian skeletal muscle, mammalian cardiac muscle, mouse dorsal root ganglion cells in vitro and in culture, cultured mammalian skeletal muscle, and L6 cells. See Rogart, Ann. Rev. Physiol. 43:711-725 (1981).

Dorsal root ganglia neurons possess both TTX-sensitive (IC₅₀ ≈0.3 nM) and TTX-resistant (IC₅₀≈100 μM) sodium channel currents, as described in Roy, et al., J. Neurosci. 12:2104-2111 (1992).

TTX-resistant sodium currents have also been measured in rat nodose and petrosal ganglia, Ikeda, et al., J. Neurophysiol. 55:527-539 (1986) and Stea, et al., Neurosci. 47:727-736 (1992).

SUMMARY OF THE INVENTION

One aspect of the present invention is a purified and isolated DNA sequence encoding for a novel TTX-sensitive sodium channel protein, in particular, the α-subunit of this protein. Another embodiment of the invention is a purified and isolated DNA sequence encoding for a splice variant of the novel TTX-sensitive sodium channel.

Another aspect of the invention is a method of stabilizing the full length cDNA which encodes the protein sequence of the invention.

Also included in this invention are alternate DNA forms, such as genomic DNA, DNA prepared by partial or total chemical synthesis from nucleotides, and DNA having deletions or mutations.

Another aspect of the invention is a novel probe based on known sodium channels for screening rat cDNA libraries.

Further aspects of the invention include expression vectors comprising the DNA of the invention, host cells transformed or transfected by these vectors, and clonal cell lines expressing the DNA of the invention. Also disclosed is the cDNA and MRNA derived from the DNA sequences of the invention.

Another aspect of the present invention are recombinant polynucleotides and oligonucleotides comprising a nucleic acid sequence derived from the DNA sequence of this invention.

Still another aspect of the invention is the novel rat TTX-sensitive sodium channel protein and fragments thereof, encoded by the DNA of this invention.

Further provided is a method of inhibiting the activity of the novel TTX-sensitive sodium channel comprising administering an effective amount of a compound having an IC₅₀ of 1 nM or less.

Also forming part of this invention is an assay for inhibitors of the sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel.

Another part of this invention is a method of employing the DNA for forming monoclonal and polyclonal antibodies, for use as molecular targets for drug discovery, highly specific markers for specific antigens, detector molecules, diagnostic assays, and therapeutic uses.

BRIEF DESCRIPTION OF THE SEQ ID'S AND FIGURES

SEQ ID NO: 1 depicts an engineered version of the nucleotide cDNA sequence encoding the rat TTX-sensitive peripheral nerve sodium channel type 4 (“PN4”). This version lacks most of the untranslated sequences, thereby comprising a 5934-base open reading frame, from nucleotide residue 22 of the XhoI-HindIII clone, the start site of translation, and ending at residue 5956.

SEQ ID NO:2 depicts an engineered version of the nucleotide cDNA sequence encoding the rat TTX-sensitive peripheral nerve sodium channel type 4a (“PN4a”). This version lacks most of the untranslated sequences, thereby comprising a 5964-base open reading frame, beginning at nucleotide residue 22 of the XhoI-HindHIII clone, the start site of translation, and ending at residue 5986. The 30 base pair insert is found at positions 2014-2043.

FIGS. 1A-1C (SEQ ID NO:3) depict the deduced amino acid sequence of PN4, represented in the single-letter amino acid code. Shown in FIG. 1A-1C are the homologous domains (I-IV); the putative transmembrane segments (S1-S6); the amino acid conferring sensitivity to TTX (Δ); potential cAMP-phosphorylation site (•); and potential N-glycosylation site (♦).

FIGS. 2A-2C (SEQ ID NO:4) depict the deduced amino acid sequence of PN4a, represented in the single-letter amino acid code. Shown in FIGS. 2A-2C are the homologous domains (I-IV); the putative transmembrane segments (S1-S6); the amino acid conferring sensitivity to TTX (Δ); potential cAMP-phosphorylation site (•); and potential N-glycosylation site (♦).

FIGS. 3A-3H (SEQ ID NOS:7 and 8 align the base pair sequences of the NaCh6 and the “native” version of the PN4 sodium channel cDNA clones (SEQ ID NOS:7 and 8, including untranslated sequences, depicting the differences in bold. Start and stop codons are underlined and primers are denoted by dashed lines with arrows.

FIGS. 4A-4D align the amino acid sequences of the PN4a (SEQ ID NO:4), PN4(SEQ ID NO:3), and NaCh6 sodium channel (SEQ ID NO:9) cDNA clones of FIGS. 3A-3H, depicting the differences in bold.

FIG. 5 is a comparison of the conserved region Interdomain I/II between PN4a (SEQ ID NO:10), PN4(SEQ ID NO:11), NaCh6(SEQ ID NO:12), and BrainII sodium channels (SEQ ID NO:13). Differences between PN4 (and PN4a) and NaCh6 are shown in bold type and differences between BrainII and PN4 are underlined.

SEQ ID NO:5 depicts the 696 nucleotide cDNA sequence encoding the novel probe CNaD4-2 used to identify the novel sodium channels of the invention.

SEQ ID NO:6 depicts the deduced amino acid sequence of probe CNaD4-2, represented in the single-letter amino acid code.

FIG. 6 depicts the cloning map of PN4 and PN4a.

FIG. 7 shows the properties of currents produced in Xenopur oocytes by injection of PN4 cRNA. FIG. 7a shows the current produced by sodium channels expressed in an oocyte; FIG. 7b shows the current-voltage relationship.

FIGS. 8a and 8 b show steady state inactivation of sodium currents produced by PN4 in Xenopur oocytes .

FIG. 9 demonstrates the effects of the sodium channel β₁ and β₂ subunits upon PN4 function in Xenopur oocytes . FIG. 9a shows currents produced when the PN4I subunit is injected alone; FIG. 9b is with the β₁ subunit; FIG. 9c is with the β₂ subunit; and FIG. 9d is with both the β₁ and β₂ subunits.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel sodium channel proteins. Specific embodiments include the α-subunit of such sodium channels that are TTX-sensitive.

In particular, the present invention relates to a purified and isolated DNA sequence encoding for a novel rat TTX-sensitive sodium channel protein and a splice variant thereof. The term “purified and isolated DNA” refers to DNA that is essentially free, i.e. contains less than about 30%, preferably less than about 10%, and more preferably less than about 1% of the DNA with which the DNA of interest is naturally associated. Techniques for assessing purity are well known to the art and include, for example, restriction mapping, agarose gel electrophoresis, and CsCl gradient centrifugation. The term “DNA” is meant to include cDNA made by reverse transcription of MRNA or by chemical synthesis.

Specifically, the invention encompasses DNA having the engineered versions (discussed in detail below) of the nucleotide sequences set forth in SEQ ID NOS:1 and 2 designated herein as nerve sodium channel types 4 and the splice variant 4a (PN4 and PN4a). These versions of the PN4 and PN4a sequence were produced by removing most of the untranslated sequences of the PN4 and PN4a cDNA and cloning into expression vectors for functional analysis.

The longer “native” version of PN4 is shown in FIGS. 3A-3H (SEQ ID NO:7). The complete “native” base pair sequence of PN4a has the same sequence shown in FIG. 3 and is labeled rPN4 with the 30 base pair insert after position 2050. The PN4 and PN4a DNA sequences comprise cDNA sequences that encode the α-subunit of novel voltage-gated, TTX-sensitive sodium channels, specifically the amino acid sequences set forth in FIGS. 1A-1C and 2 (SEQ ID NOS:3 and 4). DNA sequences encoding the same or allelic variant or analog sodium channel protein polypeptides of the nervous system, through use of, at least in part, degenerate codons are also contemplated by this invention.

The nucleotide sequences of SEQ ID NOS:1 and 2 correspond to the cDNAs from rat. PN4 shares greater than 99% homology with the rat sodium channel NaCh6, previously cloned from brain cDNA (Schaller, et al., J. Neurosci. 15:3231-3242 (1995)), and also greater than 99% homology with the orthologous mouse channel, Scn8a (Burgess, et al., Nature Genetics 10:61-465 (1995)), discounting the 738 bp deletion in the interdomain I/II region of Scn8a relative to PN4. A homology search provided that the next closest related sodium channel is found in the fugu (puffer fish), with 92% homology. The next closest channels are rat brain types I and II, at 87.9%, and rat brain type III, at 87.3%. Homology to all other known channels drops off significantly thereafter.

Additionally, it is believed that the novel voltage-gated, TTX-sensitive sodium channel is also expressed in tissue of other mammalian species such as humans, and that the corresponding gene is highly homologous to the rat sequence. Therefore, the invention includes cDNA encoding a novel mammalian voltage-gated, TTX-sensitive sodium channel.

The invention not only includes the entire protein expressed by the cDNA sequences of SEQ ID NOS:1 and 2, but also includes protein fragments. These fragments can be obtained by cleaving the full length proteins or by using smaller DNA sequences or polynucleotides to express the desired fragment. Accordingly, the invention also includes polynucleotides that can be used to make polypeptides of about 10 to 1500, preferably 10 to 100, amino acids in length. The isolation and purification of such recombinant polypeptides can be accomplished by techniques that are well known in the art, for example, preparative chromatographic separations or affinity chromatography. In addition, polypeptides can also be made by synthetic means which are well known in the art.

In general, sodium channels comprise an α- and one or more β-subunits. The β-subunits may modulate the function of the channel. However, since the β-subunit is all that is required for the channel to be fully functional, expression of the cDNA in SEQ ID NOS:1 and 2 will each provide a fully functional protein. The gene encoding the β₁-subunit in nerve tissue was found to be identical to that found in rat heart, brain, and skeletal muscle. The cDNA of the β₁-subunit is not described herein as it is well known in the art, see Isom, et al., Neuron 12:1183-1194 (1994). However, it is to be understood that by combining the known sequence for the β₁-subunit with the α-subunit sequence described herein, one may obtain complete PN4 and PN4a rat voltage-gated, TTX-sensitive sodium channels.

Northern blot analysis indicates that PN4 and PN4a are each encoded by a˜7.5 kb/9.5 kb transcript. The nucleotide sequence analysis of the PN4 cDNA identifies a 5934-base open reading frame, shown in SEQ ID NO: 1, starting at base 22. The nucleotide sequence analysis of the PN4a cDNA identifies a 5964-base open reading frame, shown in SEQ ID NO:2, also starting at base 22. The deduced amino acid sequence of PN4, shown in FIGS. 1A-1C (SEQ ID NO:3), exhibits the primary structural features of an α-subunit of a voltage-gated, TTX-sensitive sodium channel. Shown in FIG. 1A-1C are the homologous domains (I-IV); the putative transmembrane segments (S1-S6); the amino acid conferring sensitivity to TTX (Δ); potential cAMP-phosphorylation site (•); and potential N-glycosylation site (♦). The deduced amino acid sequence of PN4a, shown in FIGS. 2A-2C (SEQ ID NO:4), also exhibits the primary structural features of an α-subunit of a voltage-gated, TTX-sensitive sodium channel. Shown in FIG. 2A-2C are the homologous domains (I-IV); the putative transmembrane segments (S1-S6); the amino acid conferring sensitivity to TTX (Δ); potential cAMP-phosphorylation site (•); and potential N-glycosylation site (♦).

Reverse transcription-polymerase chain reaction (degenerate oligonucleotide-primed “RT-PCR”) analysis of RNA from the rat central and peripheral nervous systems, in particular from rat dorsal root ganglia (“DRG”), was performed. Eight main tissue types were screened by f5 RT-PCR for expression of the unique PN4 genes corresponding to positions 4646-5203 of SEQ ID NO:1. PN4 was present in five of the tissues studied: brain, spinal cord, DRG, nodose ganglia, and superior cervical ganglia. PN4 was not present in the remaining tissues studied: sciatic nerve tissue, heart tissue, or skeletal muscle tissue.

Three main tissue types were screened by RT-PCR for expression of the unique PN4a genes corresponding to positions 1947-2135 of SEQ ID NO:2. PN4a was present in two of the tissues studied: spinal cord and DRG. PN4a was not present in brain tissue.

The invention also pertains to the cloning and functional expression in Xenopur oocytes of the novel PN4 and PN4a rat TTX-sensitive sodium channels. Specifically, the α-subunit of the sodium channels was cloned and expressed. Functional expression shows that PN4 and PN4a are voltage-gated, TTX-sensitive sodium channels with properties that are similar to other TTX-sensitive sodium channels.

Preferred aspects of this invention are PN4 cDNA sequences which encode for the novel mammalian TTX-sensitive sodium channel proteins that are expressed in brain, spinal cord, dorsal root ganglia, nodose ganglia, and superior cervical ganglia but not in sciatic nerve, heart, or skeletal muscle when assayed by the methods described herein, such as RT-PCR.

Also preferred aspects of this invention are PN4a cDNA sequences which encode for the novel mammalian TTX-sensitive sodium channel proteins that are expressed most strongly in DRG, with little expression in spinal cord and almost undetectable expression in brain when assayed by the methods described herein, such as RT-PCR.

cDNA sequences which encode for the novel PN4 TTX-sensitive sodium channel proteins that are predominantly expressed in the brain and spinal cord are also contemplated by this invention. cDNA sequences which encode for the novel PN4a TTX-sensitive sodium channel proteins that are predominantly expressed in the DRG are also contemplated by this invention.

The term “cDNA”, or complementary DNA, refers to single-stranded or double-stranded DNA sequences obtained by reverse transcription of MRNA isolated from a donor cell. For example, treatment of MRNA with a reverse transcriptase such as AMV reverse transcriptase or M-MuLV reverse transcriptase in the presence of an oligonucleotide primer will furnish an RNA-DNA duplex which can be treated with RNase H, DNA polymerase and DNA ligase to generate double-stranded cDNA. If desired, the double-stranded cDNA can be denatured by conventional techniques such as heating to generate single-stranded cDNA. The term “cDNA” includes cDNA that is a complementary copy of the naturally occurring mRNA, as well as complementary copies of variants of the naturally occurring MRNA, that have the same biological activity. Variants would include, for example, insertions, deletions, and sequences with degenerate codons and alleles. For example, PN4a is a splice variant of PN4, having a 10 amino acid insertion.

The term “cRNA” refers to RNA that is a copy of the MRNA transcribed by a cell. cRNA corresponding to MRNA transcribed from a DNA sequence encoding the α-subunit of a novel TTX-sensitive sodium channel protein is contemplated by this invention.

The present invention also includes expression vectors comprising the DNA or the cDNA described above, host cells transformed with these expression vectors capable of producing the sodium channel of the invention, and cDNA libraries comprising such host cells.

The term “expression vector” refers to any genetic element, e.g., a plasmid, a chromosome, a virus, behaving either as an autonomous unit of polynucleotide expression within a cell or being rendered capable of replication by insertion into a host cell chromosome, having attached to it another polynucleotide segment, so as to bring about the replication and/or expression of the attached segment. Suitable vectors include, but are not limited to, plasmids, bacteriophages, and cosmids. Vectors will contain polynucleotide sequences which are necessary to effect ligation or insertion of the vector into a desired host cell and to effect the expression of the attached segment. Such sequences differ depending on the host organism and will include promoter sequences to effect transcription, enhancer sequences to increase transcription, ribosomal binding site sequences, and transcription and translation termination sequences.

The term “host cell” generally refers to prokaryotic or eukaryotic organisms and includes any transformable or transfectable organism which is capable of expressing a protein and can be, or has been, used as a recipient for expression vectors or other transferred DNA. Host cells can also be made to express protein by direct injection with exogenous cRNA translatable into the protein of interest. A preferred host cell is the Xenopus oocyte.

The term “transformed” refers to any known method for the insertion of foreign DNA or RNA sequences into a host prokaryotic cell. The term “transfected” refers to any known method for the insertion of foreign DNA or RNA sequences into a host eukaryotic cell. Such transformed or transfected cells include stably transformed or transfected cells in which the inserted DNA is rendered capable of replication in the host cell. They also include transiently expressing cells which express the inserted DNA or RNA for limited periods of time. The transformation or transfection procedure depends on the host cell being transformed. It can include packaging the polynucleotide in a virus, as well as direct uptake of the polynucleotide, such as, for example, lipofection or microinjection. Transformation and transfection can result in incorporation of the inserted DNA into the genome of the host cell or the maintenance of the inserted DNA within the host cell in plasmid form. Methods of transformation are well known in the art and include, but are not limited to, viral infection, electroporation, lipofection, and calcium phosphate mediated direct uptake.

It is to be understood that this invention is intended to include other forms of expression vectors, host cells, and transformation techniques which serve equivalent functions and which become known to the art hereto.

The term “cDNA library” refers to a collection of clones, usually in a bacteriophage, or less commonly in bacterial plasmids, containing cDNA copies of mRNA sequences derived from a donor cell or tissue.

In addition, the present invention contemplates recombinant polynucleotides, of about 15 to 20kb, preferably 10 to l5kb nucleotides in length, comprising a nucleic acid sequence derived from the DNA of the invention.

The term “polynucleotide” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modified, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide.

The term “derived from” a designated sequence, refers to a nucleic acid sequence that is comprised of a sequence of approximately at least 6-8 nucleotides, more preferably at least 10-12 nucleotides, and, even more preferably, at least 15-20 nucleotides that correspond to, i.e., are homologous or complementary to, a region of the designated sequence. The derived sequence is not necessarily physically derived from the nucleotide sequence shown, but may be derived in any manner, including for example, chemical synthesis or DNA replication or reverse transcription, which are based on the information provided by the sequences of bases in the region(s) from which the polynucleotide is derived.

Further, the term “polynucleotide” is intended to include a recombinant polynucleotide, which is of genomic, cDNA, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation is not associated with all or a portion of the polynucleotide with which it is associated in nature and/or is linked to a polynucleotide other than that to which it is linked in nature.

The “native” version of PN4, and its splice variant PN4a, partially correspond to the sodium channel NaCh6, described in Schaller, K. L. et al., J. Neurosci. 15(5):3231-3242 (1995) as shown in FIGS. 3 and 4. In FIG. 3, base pair sequences of the native PN4 and PN4a sodium channel cDNA clones include untranslated sequences. The applicants believe that the published NaCh6 sequence does not correctly provide the sodium channel sequence, and that the sequences for PN4 and PN4a represent the authentic sodium channel sequence for the following reasons.

First, most sodium channel gene coding regions, including PN4, begin with an eleven base pair sequence consisting of an out of frame ATG, followed by five base pairs downstream, followed by the ATG initiation codon for the coding region. The DNA sequence alignment (FIG. 3) shows a two base pair deletion in NaCh6 overlapping the second ATG, so that the normally out of frame, upstream ATG becomes the NaCh6 initiation codon, leading to a two amino acid insertion. Start and stop codons are underlined and primers are denoted by dashed lines with arrows.

Examination of the DNA sequence alignment (FIG. 3) shows that the bulk of the differences (residues in bold print) between the two sequences that would strongly influence protein function consist of a series of nine single base deletions in the Interdomain I/II region These differences lead to a very different amino acid sequence, as shown in the amino acid alignment of FIGS. 4A-4D, where the differences between the two sequences are again shown in bold print. The applicants' sequencing of multiple isolates resulting from the cloning of up to 1.5kb of the Interdomain I/II region by PCRrepeatedly resulted in sequences which completely agreed with PN4 or PN4a sequences.

Comparison of PN4 and PN4a sequences to other sodium channel sequences shows a high degree of homology. For example, FIG. 5 is a comparison of PN4 and PN4a with NaCh6 and rat Brain type II in this region. Whereas PN4 and BrainII share about 50% identity in the region highlighted in bold, NaCh6 is almost completely different. The differences between BrainII and PN4 are underlined.

The applicants also employed PCR to look specifically for NaCh6. A sense primer common to both sequences (CAATCGTGGGCGCCCTAATC (SEQ ID NO:14), corresponding to base pair 722-742 of NaCh6 and shown by dashed lines with an arrow in FIGS. 3A-3H at bases 884-904 of PN4) was paired with gene specific antisense primers (TGCTTTCATGCACTGGAATCCCTCT (SEQ ID NO:15), corresponding to base pair 1194-1170 of PN4, and TGCTTTACTGCACTGGAATCCTTCG (SEQ ID NO:10), fs corresponding to base pair 1029-1005 of NaCh6; sequence differences between the two primers are underlined). The antisense primers prime at a three base pair deletion of NaCh6 relative to PN4 and overlap three other sequence differences, as shown in FIGS. 3A-3H. A PCR product of the expected size (about 300 base pairs) was obtained with the PN4 specific antisense primer using pBK-CMV/75-1.4 DNA (described in the description of SEQ ID NO:2) and with rat Brain and rat DRG cDNA templates. No PCR products were obtained from these templates with the NaCh6 specific primer.

Any of the sequence differences between PN4 and NaCh6 could result in an inability of the NaCh6 gene to form a functional channel. However, some differences could be ascribed to “base calling.” The applicants have repeatedly sequenced full length versions of PN4 and PN4a to verify the accuracy of the sequence. Of the amino acid differences between PN4 and NaCh6, it appears that the profound differences in the Interdomain I/II region are responsible for the lack of success in expression of the NaCh6 gene. The nine single base deletions in this region appear to shift the reading frame (see FIGS. 3A-3H and FIGS. 4A-4D), leading to a “nonsense” peptide which lacks a number of highly conserved residues (FIG. 5) and which could sufficiently disrupt the structure of the protein to destroy its function.

The splice variant PN4a is similar to and occurs in a homologous position to that seen with rat type Brainl and la channels (Schaller, K. L, et al., J. Neurosci 12:1370-1381 (1992)). In each case, it appears that the variants make use of the same 3′ splice acceptor sites but alternative 5′sites. Rat BrainIII also has splice variants in this region, using the same 3′ splice site but using alternative 5′ sites more 5′ than the other channels. An amino acid comparison with other rat(r) and human(h) channels is shown below. Not all sodium channels have this splicing pattern.

rPN4 GRLLPE ........... AT.TEVE (SEQ ID NO:17) rPN4a GRLLPE VKIDKAAT.DS AT.TEVE (SEQ ID NO:18) rBRAIN1 GQLLPE VIIDKPATDDN GTTTETE (SEQ ID NO:19) rBRAIN1a GQLLPE ........... GTTTETE (SEQ ID NO:20) rPN1 GQLLPE VIIDKATSDDS GTTNQIH (SEQ ID NO:21) hNE-Na GQLLPE ........... GTTNQIH (SEQ ID NO:22) rBRAIN2 GQLLPE ........... GTTTETE (SEQ ID NO:23) rBRAIN3 ...... ........... GTTTETE (SEQ ID NO:24) rCARDIAC SYLLRP MVLDRPP..DT TTPSEEP (SEQ ID NO:25)

It is interesting to note that the species of rat PN 1 is similar to PN4a in this location, whereas its human orthologue, the neuroendocrine channel, hNE-Na (Klugbauer, et al., EMBO J. 14:1084-1090 (1995)), is similar to PN4. Perhaps each of these will be found to be one of a set of splice variants. Whereas the splicing patterns of BrainI, IL and III were found not to vary across a range of tissues (Schaller, K.L., et al., J. Neurosci. 12:1370-1381 (1992)), PN4 and PN4a show dramatic abundance differences. PN4 has a gradient of expression with high expression in brain, intermediate in spinal cord, and relatively the least in DRG. PN4a is very low or undetectable in brain, a minor fraction of total PN4 expression in spinal cord, and nearly as abundant as PN4 in DRG.

Uses of the Invention

Many uses of the invention exist, a few of which are described below.

1. Probe for human channel.

As mentioned above, it is believed that homologs of the novel rat TTX-sensitive sodium channel described herein are also expressed in mammalian nerve tissue, in particular, human tissue. The entire cDNAs of PN4 and PN4a rat sodium channels of the present invention can be used as a probe to discover whether novel PN4 and PN4a voltage-gated, TTX-sensitive sodium channels exist in human nerve tissue and, if they do, to aid in isolating the cDNAs for the human protein.

The human homologues of the rat TTX-sensitive PN4 and PN4a channels can be cloned using a human DRG cDNA library. Human DRG are obtained at autopsy. The frozen tissue is homogenized and the RNA extracted with guanidine isothiocyanate (Chirgwin, et al., Biochernistry 18:5294-5299, 1979). The RNA is size-fractionated on a sucrose gradient to enrich for large mRNAs because the sodium channel α-subunits are encoded by large (7-11 kb) transcripts. Double-stranded cDNA is prepared using the SuperScript Choice cDNA kit (GIBCO BRL) with either oligo(dT) or random hexamer primers. EcoRI adapters are ligated onto the double-stranded cDNA, which is then phosphorylated. The cDNA library is constructed by ligating the double-stranded cDNA into the bacteriophage-lambda ZAP II vector (Stratagene) followed by packaging into phage particles.

Phage are plated out on 150 mm plates on a lawn of XLI-Blue MRF bacteria (Stratagene) and plaque replicas are made on Hybond N nylon membranes (Amersham). Filters are hybridized to rat PN4 and PN4a cDNA probes by standard procedures and detected by autoradiography or chemiluminescence. The signal produced by the rat PN4 and PN4a probes hybridizing to positive human clones at high stringency should be stronger than obtained with rat brain sodium channel probes hybridizing to these clones. Positive plaques are further purified by limiting dilution and re-screened by hybridization or PCR. Restriction mapping and polymerase chain reaction will identify overlapping clones that can be assembled by standard techniques into the full-length human homologue of rat PN4 and PN4a. The human clone can be expressed by injecting cRNA transcribed in vitro from the full-length cDNA clone into Xenopur oocytes , or by transfecting a mammalian cell line with a vector containing the cDNA linked to a suitable promoter.

2. Probe for Obtaining Molecular Data.

The polynucleotides of the invention can be bound to a reporter molecule to form a polynucleotide probe useful for Northern and Southern blot analysis and in situ hybridization.

The term “reporter molecule” refers to a chemical entity capable of being detected by a suitable detection means, including, but not limited to, spectrophotometric, chemiluminescent, immunochemical, or radiochemical means. The polynucleotides of this invention can be conjugated to a reporter molecule by techniques well known in the art. Typically the reporter molecule contains a functional group suitable for attachment to or incorporation into the polynucleotide. The functional groups suitable for attaching the reporter group are usually to activated esters or alkylating agents. Details of techniques for attaching reporter groups are well known in the art. See, for example, Matthews, J. A., Batki, A., Hynds, C., and Kricka, L. J., Anal. Biochem., 151:205-209 (1985) and Engelhardt, et al., European Patent Application No. 0 302 175.

3. Antibodies Against PN4 and PN4a.

The polypeptides of the invention are highly useful for the development of antibodies against PN4 and PN4a. Such antibodies can be used in affinity chromatography to purify recombinant sodium channel proteins or polypeptides, or they can be used as a research tool. For example, antibodies bound to a reporter molecule can be used in histochemical staining techniques to identify other tissues and cell types where PN4 and PN4a are present, or they can be used to identify epitopic or functional regions of the sodium channel protein of the invention.

The antibodies can be monoclonal or polyclonal and can be prepared by techniques that are well known in the art. Polyclonal antibodies are prepared as follows: an immunogenic conjugate comprising PN4, PN4a, or a fragment thereof, optionally linked to a carrier protein, is used to immunize a selected mammal such as a mouse, rabbit, goat, etc. Serum from the immunized mammal is collected and treated according to known procedures to separate the immunoglobulin fraction.

Monoclonal antibodies are prepared by standard hybridoma cell technology based on that reported by Kohler and Milstein in Nature 256:495-497 (1975): spleen cells are obtained from a host animal immunized with the PN4 or PN4a protein or a fragment thereof, optionally linked to a carrier. Hybrid cells are formed by fusing these spleen cells with an appropriate myeloma cell line and cultured. The antibodies produced by the hybrid cells are screened for their ability to bind to expressed PN4 or PN4a proteins.

A number of screening techniques well known in the art, such as, for example, forward or reverse enzyme-linked immunosorbent assay screening methods may be employed. The hybrid cells producing such antibodies are then subjected to recloning and high dilution conditions in order to select a hybrid cell that secretes a homogeneous population of antibodies specific to either the PN4 or PN4a protein.

In addition, antibodies can be raised by cloning and expressing nucleotide sequences or mutagenized versions thereof coding at least for the amino acid sequences required for specific binding of natural antibodies, and these expressed proteins used as the immunogen. Antibodies may include the complete immunoglobulin or a fragment thereof. Antibodies may be linked to a reporter group such as is described above with reference to polynucleotides.

4. Therapeutic Targets for Disorders.

The present invention also includes the use of the novel voltage-gated, TTX-sensitive sodium channel α-subunit as a therapeutic target for compounds to treat disorders of the nervous system including, but not limited to, epilepsy, stroke injury, brain injury, allodynia, hyperalgesia, diabetic neuropathy, traumatic injury, and AIDS-associated neuropathy. The invention allows for the manipulation of genetic materials by recombinant technology to produce polypeptides that possess the structural and functional characteristics of the novel voltage-gated, TTX-sensitive sodium channel α-subunit found in nerve tissue, particularly in sensory nerves. Site directed mutagenesis can be used to provide such recombinant polypeptides. For example, synthetic oligonucleotides can be specifically inserted or substituted into the portion of the gene of interest to produce genes encoding for and expressing a specific mutant. Random degenerate oligonucleotides can also be inserted and phage display techniques can be used to identify and isolate polypeptides possessing a functional property of interest.

5. Designing Therapeutics based on Inhibiting PN4 and PN4a and Assays thereof.

This invention is also directed to inhibiting the activity of PN4 in brain, spinal cord, DRG, nodose ganglia, and superior cervical ganglia tissues. This invention is also directed to inhibiting the activity of PN4a in spinal cord and DRG tissues. However, it is to be understood that further studies may reveal that PN4 and PN4a are present in other tissues, and as such, those tissues can also be targeted areas. For example, the detection of PN4 mRNA in nodose ganglia suggests that PN4 may conduct TTX-sensitive sodium currents in this and other sensory ganglia of the nervous system.

In addition, it has been found that proteins not normally expressed in certain tissues are expressed in a disease state. Therefore, this invention is intended to encompass the inhibition of PN4 and PN4a in tissues and cell types where the protein is normally expressed, and in those tissues and cell types where the protein is only expressed during a disease state.

The invention also pertains to an assay for inhibitors of the novel TTX-sensitive sodium channel protein comprising contacting a compound suspected of being an inhibitor with expressed sodium channel and measuring the activity of the sodium channel. The compound can be a substantially pure compound of synthetic origin combined in an aqueous medium, or the compound can be a naturally occurring material such that the assay medium is an extract of biological origin, such as, for example, a plant, animal, or microbial cell extract. PN4 and PN4a activity can be measured by methods such as electrophysiology (two electrode voltage clamp or single electrode whole cell patch clamp), guanidinium ion flux assays, and toxin-binding assays. An “inhibitor” is defined as generally that amount that results in greater than 50% decrease in PN4 or PN4a activity, preferably greater than 70% decrease in PN4 or PN4a activity, more preferably greater than 90% decrease in PN4 or PN4a activity.

6. Designing and Screening for Additional Therapeutics.

Another significant characteristic of PN4 is that it is TTX-sensitive. It is believed that TTX-sensitive sodium channels play a key role in transmitting nerve impulses relating to sensory inputs such as pain and pressure. This will also facilitate the design of therapeutics that can be targeted to a specific area such as nerve tissue.

Additionally, the recombinant protein of the present invention can be used to screen for potential therapeutics that have the ability to inhibit the sodium channel of interest. In particular, it would be useful to inhibit selectively the function of sodium channels in nerve tissues responsible for transmitting pain and pressure signals without simultaneously affecting the function of sodium channels in other tissues such as heart and muscle. Such selectivity would allow for the treatment of pain without causing side effects due to cardiac or neuromuscular complications. Therefore, it would be useful to have DNA sequences coding for sodium channels that are selectively expressed in nerve tissue.

7. Pain Reliever.

Sodium channels in nerve tissue play a large role in the transmission of nerve impulses, and therefore are instrumental in understanding neuropathic pain transmission. Neuropathic pain falls into two categories: allodynia, where a normally non-painful stimulus becomes painful, and hyperalgesia, where a usually normal painful stimulus becomes extremely painful. The ability to inhibit the activity of these sodium channels, i.e., reduce the conduction of nerve impulses, will affect the nerve's ability to transmit pain. Selective inhibition of sodium channels in sensory neurons such as dorsal root ganglia will allow the blockage of pain impulses without complicating side effects caused by inhibition of sodium channels in other tissues such as brain and heart. In addition, certain diseases are caused by sodium channels that produce impulses at an extremely high frequency. The ability to reduce the activity of the channel can then eliminate or alleviate the disease. Accordingly, potential therapeutic compounds can be screened by methods well known in the art, to discover whether they can inhibit the activity of the recombinant sodium channel of the invention. Barram, M., et al., Naun-Schmiedeberg's Archives of Pharmacology, 347:125-132 (1993) and McNeal, E. T., et al., J. Med. Chem., 28:381-388 (1985). For similar studies with the acetyl choline receptor, see, Claudio, et al., Science, 238:1688-1694 (1987).

Accordingly, the present invention encompasses a method of alleviating pain by inhibiting the activity of the novel TTX-sensitive sodium channel comprising administering a therapeutically effective amount of a compound having an IC₅₀ in the range of 0.1-50 nM, preferably within the range of 1-25 nM, and more preferably within the range of 1-5 nM. Potential therapeutic compounds are identified based on their ability to inhibit the activity of PN4 and PN4a. Therefore, the aforementioned assay can be used to identify compounds having a therapeutically effective IC₅₀.

The term “IC₅₀” refers to the concentration of a compound that is required to inhibit by 50% the activity of expressed PN4 or PN4a when activity is measured by electrophysiology, flux assays, and toxin-binding assays, as mentioned above.

The basic molecular biology techniques employed in accomplishing features of this invention, such as RNA, DNA, and plasmid isolation, restriction enzyme digestion, preparation and probing of a cDNA library, sequencing clones, constructing expression vectors, transforming cells, maintaining and growing cell cultures, and other general techniques are well known in the art, and descriptions of such techniques can be found in general laboratory manuals such as Molecular Cloning: A Laboratory Manual by Sambrook, et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989). Accordingly, the following examples are merely illustrative of the techniques by which the invention can be practiced.

Abbreviations BSA bovine serum albumin Denhardt's 0.02% BSA, 0.02% polyvinyl-pyrrolidone, 0.02% Ficoll solution (0.1 g BSA, 0.1 g Ficoll and 0.1 g polyvinylpyrrolidone per 500 ml) DRG dorsal root ganglia EDTA Ethylenediaminetetraacetic acid, tetrasodium salt MEN 20 mM MOPS, 1 mM EDTA, 5 mM sodium acetate, pH 7.0 MOPS 3-(N-morpholino)propanesulfonic acid (Sigma Chemical Company) PN3 peripheral nerve sodium channel type 3 PNS peripheral nervous system SDS sodium dodecyl sulfate SSC 150 nM NaCl, 15 mM sodium citrate, pH 7.0 SSPE 80 mM NaCl, 10 mM sodium phosphate, 1 mM ethylenediaminetetraacetate, pH 8.0 TEV two electrode voltage clamp TTX tetrodotoxin (Sigma Chemical Company) UTR untranslated region

EXAMPLES

Each step employed in obtaining the DNA of the novel sodium channel of the invention is described in the detailed examples below. The following is an overview of the steps. Example 1 describes how a novel probe, CNaD4-2, was obtained by designing primers based on known sodium channels. Example 2 describes the construction and screening of a cDNA library with CNaD4-2 to obtain the 3′ end of the novel sodium channel of the invention. Then a known primer was employed to obtain the 5′ end of the DNA of the invention. Example 3 describes how RT-PCR was employed to span the gap, between the 3′ and 5′ ends obtained from the cDNA library. This resulted in a 798 base pair sequence and a splice variant thereof, having a 828 base pair sequence. Example 4 describes assembling the clones into two full-length cDNA clones in expression vectors. The cloning map is illustrated in FIG. 6. Example 5 discusses the tissue distribution and localization accomplished by RT-PCR. Example 6 discusses the northern analysis of mRNA. Example 7 discloses obtaining expression data from Xenopur oocytes , and localization by RT-PCR.

Materials

The plasmid pBK-CMV was obtained from Stratagene (La Jolla, Calif.); plasmid Litmus 29 was obtained from New England Biolabs (Beverly, Mass.); the oocyte expression vector plasmid pBSTAcIIr was constructed from pBSTA (obtained from A. Goldin at the University of California, Irvine and described by Goldin, et al., in Methods in Enzymology (Rudy & Iverson, eds.) 207:279-297) by insertion of a synthetic oligonucleotide linker; the mammalian cell expression vector plasmid pCI-neo was obtained from Promega (Madison, Wis.); plasmid pCRII was obtained from Invitrogen (San Diego, Calif.). Competent E. Coli cell lines STBL2TM and SURE® were obtained from GIBCO/BRL and Stratagene, respectively.

EXAMPLE 1

Identification of a novel channel fragment

A novel probe used to identify the novel sodium channels was obtained as follows. Degenerate oligonucleotide primers were designed based on the homologies between known sodium channels in domain IV and used to perform RT-PCR on RNA isolated from rat DRG. The domain IV PCR products were cloned into pCRII, transformed into E. coli, and single colonies isolated. DNA sequence of the inserts of several of these colonies was obtained, including the following novel sequence from clone pCRII/CNaD4-2 of SEQ ID NO:5, identified as CNaD4-2. SEQ ID NO:6 depicts the deduced amino acid sequence of probe CNaD4-2, represented in the single-letter amino acid code.

CNaD4-2 can be made with standard PCR techniques.

EXAMPLE 2

Construction and screening of cDNA library from rat DRG with probe CNaD4-2

EcoRI-adapted cDNA was prepared from normal adult male Sprague-Dawley rat DRG poly(A)+ RNA using the SuperScript Choice System (GIBCO BRL). cDNA (>4kb) was selected by sucrose gradient fractionation as described by Kieffer, Gene 109:115-119 (1991). The cDNA was then ligated into the Zap Express vector (Stratagene) and packaged with the Gigapack II XL lambda packaging extract (Stratagene). Plate lysates were prepared and screened by PCR using CNaD4-2 specific primers (ACACTCAGAGCAAGCAGATGG (SEQ ID NO: 26) and TCCCTGGGTGCTCTTTGTCCA (SEQ ID NO: 27), corresponding to bases 32 to 52 and 569 to 589 of SEQ ID NO:5, respectively). Phage from one positive lysate were screened by filter hybridization with a ³²P-labeled probe (the 700 base pair EcoRI insert from CNaD4-2). Filters were hybridized in 50% formamide, 5× SSPE, 5× Denhardt's solution, 0.5% SDS, 250 μg/ml sheared, denatured salmon sperm DNA, and 5OmM sodium phosphate at 42° C. and washed in 0.5×SSC, 0.1% SDS at 50° C. Positive clones were excised in vivo into pBK-CMV using the ExAssist/XLOLR system (Stratagene).

Approximately 95% of these clones contained sodium channel sequence under standard screening stringency conditions. The number of clones that are retrieved that contain sodium channel sequence can be increased with increased stringency conditions and careful analysis and interpretation of data. It is well known in the art when screening for a particular type of DNA sequence, other types of DNA sequences will also be hybridized, depending on the specificity of the probe. Here, with the careful designed probe of the invention, the approximate 95% “hit” rate makes this fragment an exceptionally good sodium channel probe.

One of these clones, pBK-CMV/PN4.10-1, contained sequence of the CNaD4-2 channel from domain II through the 3′ UTR. The position of the pBK-CMV/PN4.10-1 fragment in the PN4 and PN4a cloning map is shown in FIG. 6. In FIG. 6, ATG is the start codon, TAG is the stop codon and ∇ is the position of the PN4a splice insert.

A degenerate primer designed for sodium channels in domain I (ACCAACTG[T/C]GT[G/A]TT[T/C]ATGAC (SEQ ID NO:28)) was paired with a PN4 specific primer from the domain II region of pBK-CMV/PN4.10-1 (CAGCAGCTACAGTGGCTACA (SEQ ID NO:29)). These primers amplified a ca 1.5kb fragment from rat brain and from rat DRG which was shown by sequencing to represent much of the 5′ end of PN4, thus verifying that the primers would work for screening the library. The primers were then used to screen plate lysates of the DRG cDNA library by PCR. Positive lysates were plated and individual plaques picked and screened by PCR using the same primers. Positive clones were excised in vivo into pBK-CMV using the ExAssist/XLOLR system. One of these, pBK-CMV/75-1.4, was found to contain PN4 sequence from the 5′ UTR to the interdomain I/II region, but not to domain II, possibly due to rearrangement during the excision process. The position of the pBK-CMV/75-1.4 fragment in the PN4 and PN4a cloning map is shown in FIG. 6.

EXAMPLE 3

Cloning the interdomain I/II region

The gap between pBK-CMV/75-1.4 and pBK-CMV/PN4.10-1 was cloned by RT-PCR on rat DRG and brain total RNA using specific primers: AAAGAGGCCGAGTTCAAGGC (SEQ ID NO:30) (a base pair sequence of pBK-CMV/75-1.4) and TGTCCTTCCGTCCGTAGG (SEQ ID NO:31) (a base pair sequence of pBK-CMV/PN4.10-1). PCR products were cloned into plasmid pCRII and sequenced. Two distinct sequences, FA-2 and FA-7 (see FIG. 6), were cloned from DRG. These were found to be identical except for the presence of a 30 base pair insert (found at base pairs 2014-2043 in SEQ ID NO:2 and depicted by an upside triangle at the position of insertion in FA-7, FJ-13, and PN4a in FIG. 6), with sequence identity to pBK-CMV/75-1.4 and pBK-CMV/PN4.10-1 in the regions where they overlap. RT-PCR on rat brain RNA yielded only clones which lacked the 30 base pair insert. This insert is homologous to a splice variant of the NaChI channel (NaChIa) and likely results from alternative 5′ splice site usage (Schaller, K. L., et al., J. Neurosci. 12:1370-1381 (1992)).

Additional RT-PCR was performed on rat DRG RNA using primers TTCATGGGGAACCTTCGAAAC (SEQ ID NO:32) (a base pair sequence of pBK-CMV/75-1.4) and GAACGATGCAGATGGTGATGGCTAA (SEQ ID NO:33) (a base pair sequence of pBK-CMV/PN4.10-1). The 1.5 kb PCR product was cloned into pCRII; six out of twenty isolates were positive for the 30 base pair insert variant by PCR. The sequence obtained for one of these, FJ-13, position shown in FIG. 6, was identical to that expected from the sequences of pBK-CMV/75-1.4, FA-7, and pBK-CMV/PN4. 10-1, thus confirming that these clones all originated from the same transcript.

EXAMPLE 4

Assembly of full-length PN4 clones in expression vectors

Unsuccessful attempts have been made to create and stabilize full-length sodium channel cDNA sequences. In U.S. Pat. No. 5,380,836, the cDNA sequence for a rat cardiac sodium channel protein was contained in three separate plasmids. In order to create full-length functional PN4 genes, the 5′ end was modified: suitable restriction sites were added and the upstream out-of-frame initiation codon was removed. The modified pBK-CMV75-1.4 and FA-2 sequences were fused together, then combined with the remaining portion of PN4 from pBK-CMV/PN4.10-1 in suitable expression vectors. PCR was employed to assemble the 5′ portion of PN4 from the initiation codon to domain II. A 1.43kb PCR fragment was generated from pBK-CMV/75-1.4 using the following primers: (1)

GAAGCTCGAGCCCGGGCAAGAGAAGATGGCAGCGCGG (SEQ ID NO:34) (Xho-I Srf-I restriction sites underlined, initiation codon in bold, PN4 homology in italics, a base pair sequence of pBK-CMV/75-1.4) and primer (2) CTCGGAGAGCCTACCCCATC (SEQ ID NO:35) (a base pair sequence of pBK-CMV/75-1.4 and a base pair sequence of FA-2). A 0.69 kb PCR fragment was generated from FA-2 using primer (3) AGAAGGGGAAGATGGGGTAGG (SEQ ID NO:36) (a base pair sequence of FA-2 and a base pair sequence of pBK-CMV/75-1.4) and primer (4) ATTCTGTCCTTCCGTCCGTAG (a base pair sequence of FA-2 and a base pair sequence of pBK-CMV/PN4.10-1). These fragments were gel purified and then a small fraction of each was combined as template in a further PCR reaction using primers (1) and (4). The fragments share a 31 base pair region of overlap at their 3′ and 5′ ends respectively, and therefore can act as primers to fuse the two fragments together (Horton, R. M., et al. (1991) Gene 77:61-68). The 2.1kb PCR product was cloned into pCRII and several isolates were sequenced, one of which, FD-8, had the expected sequence. The position of FD-8 in the PN4 and PN4a clones is shown in the cloning map of FIG. 6.

To facilitate cloning into pBSTA and pCI-neo, it was determined to introduce an XbaI site at the 3′ end. To accomplish this, the PN4 domain II to 3′ UTR region was subcloned from pBK-CMV/PN4.10-1 from the EcoRI site of the vector to the HindIII site 14 base pairs from the PN4 stop codon into EcoRI plus HindIII digested Litmus 29. The resulting clone was labeled FC-1. The position of FC-1 in the PN4 and PN4a clones is shown in the cloning map of FIG. 6.

To assemble the full length PN4, the 5′ portion was subcloned from FD-8 as a 2.0 kb Xho I-Eco NI fragment together with the 3′ portion from FC-1 as a 4.0 kb Eco NI-Xba-I fragment into Xho-I plus Xba-I digested pBSTAcIIr. One of the resulting isolates was found to have the correct sequence and was named pBSTAcII_PN4(FU-7A).

The splice variant, PN4a, was assembled by replacing the 1.3kb Sph I—Acc I region of pBSTAcIIr_PN4(FU-7A) with the corresponding fragment from FJ-13, to form pBSTAcIIr_PN4a(FZA-3), and confirmed by DNA sequencing

PN4 and PN4a were recloned into pCI-neo as 6.0 kb Xho-I to Xba-I fragments to form pCI-neo-PN4(GAII-1) and pCI-neo-PN4a(GCII-2), respectively, and confirmed by DNA sequencing. The sequences of the coding regions as cloned in the oocyte and mammalian cell expression vectors of PN4 and PN4a are SEQ ID NO:1 and SEQ ID NO:2, respectively.

Growth of fragments of PN4 or PN4a was accomplished under standard conditions; however, growth of plasmids containing full length constructs of PN4 and PN4a (in pCIneo or pBSTAcIIr) could not be accomplished without use of special growth media, conditions, and E. coli strains. The following proved to be optimal: (1) Use of E. coli STBL2™ for primary transformation following ligation reactions; for large scale culturing the primary transformants in STBL2™ cells were used, but secondary transformants in SURE® cells were used later if necessary. These E. coli strains have altered genotypes which allow the stable propagation of plasmids containing unstable inserts. (2) Solid media was 1/2× FM (see below) plus either 1× YENB (Bacto Yeast Extract, 0.75%, Bacto Nutrient Broth, 0.8%; Sharma, R. C. and Schimike, R. T., Biotechniques 20: 42-44, 1996), 1× YET (Bacto Yeast Extract, 0.75%, Bacto Tryptone, 0.8%), or 1× LB (Tryptone, 1%, Yeast Extract, 0.5%, NaCl, 0.5%), plus 15 g/L agar. (3)Liquid media optimally was 1× FM plus 1/2× LB. (4) Carbenicillin, 100μg/ml, was used for all media, as it is metabolized less rapidly than ampicillin. However, carbenicillin may be used within the range of 50-200 μg/ml; and more preferably within the range of 75-125 μg/ml. (5) Temperature for growth should be no greater than 30° C., usually 28° C.; this necessitated longer growth periods than normally employed, from 36 to 48 hours.

The recipe for 2× Freezing Medium (2×FM) is K2HPO4, 12.6 g; Na3Citrate, 0.9 g; MgSO4.7H20, 0.18 g; (NH4)2SO4, 1.8 g; KH2PO4, 3.6 g; Glycerol, 88 g; H2O, qs to 1 L.

2×FM and the remaining media components are prepared separately, sterilized by autoclaving, cooled to at least 60° C., and added together to form the final medium. Carbenicillin is prepared at 25 mg/ml H2O and sterilized by filtration. 2×FM was first described for preparation of frozen stocks of bacterial cells (Practical Methods in Molecular Biology, Schleif, R. F. and Wensink, P. C., Springer-Verlag, New York (1981) pp201-202).

EXAMPLE 5

Tissue distribution by RT-PCR

Brain, spinal cord, DRG, nodose ganglia, superior cervical ganglia, sciatic nerve, hearts and skeletal muscle tissue were isolated from anesthetized, normal adult male Sprague-Dawley rats and were stored at −80° C. RNA was isolated from each tissue using RNAzol (Tel-Test, Inc.). Random-primed cDNA was reverse transcribed from 500 ng of RNA from each tissue. The CNaD4-2 specific primers ACACTCAGAGCAAGCAGATGG (SEQ ID NO:38) and TCCCTGGGTGCTCTTTGTCCA (SEQ ID NO:39) (see above) defined a 558 base pair amplicon and would not discriminate between PN4 and PN4a. Thermal cycler parameters were 30 s/94° C., 30 s/64° C., 1 min/72° C. (24 cycles (confirmation experiment: 34 cycles)), 30 s/94° C., 30 s/64° C., 30 s/64° C. (1 cycle). A positive control (pCRII/CNaD4-2) and a no-template control were also included. cDNA from each tissue was also PCR amplified using primers specific for glyceraldehyde-3-phosphate dehydrogenase to demonstrate template viability, as described by Tso, et al., Nucleic Acid Res. 13:2485-2502 (1985).

Tissue distribution profile of PN4 by analysis of RNA from selected rat tissues by RT-PCR was as follows:

Tissue RT-PCR (35 cycles) Brain + + + + + Spinal cord + + + DRG + + Nodose ganglia + + Superior cervical ganglia + Sciatic nerve − Heart − Skeletal muscle −

PN4 was also detected after only 25 cycles (24+1) in the same five tissues as above in the same relative abundance.

Since PN4 differs from PN4a by only 30 base pairs, a new sense primer, GGTGGACTGCAACGGCGTA (SEQ ID NO:40) (corresponding to the same base pair sequences of FA-2 and FA-7), was employed. RT-PCR using this primer together with primer ATTCTGTCCTTCCGTCCGTAG (SEQ ID NO:41) (primer 4 above) gave amplicons of 159 base pairs from PN4 and 189 base pairs from PN4a. Thermal cycler parameters were 1 min/95° C., 20sec/94° C., 30 sec/60° C., 1 min/72° C., 8 cycles, 20sec/94° C., 30 sec/58° C., 1 min/72° C., 27 cycles, 3 min/72° C. PN4a was nearly as abundant as PN4 in DRG, much less abundant than PN4 in spinal cord, and almost undetectable in brain. This correlates well with cloning data; based on sequenced, cloned RT-PCR fragments which included the 30 base pair insert region, PN4a was found in 40% of isolates from DRG (9/24), but not found from brain (0/4).

RT-PCR (35 cycles) Tissue PN4 PN4a Brain + + + + + (+/−) Spinal cord + + + + DRG + + + +

EXAMPLE 6

Northern Analysis of MRNA from rat DRG

Lumbar DRG #4 and #5 (L4 and L5), brain, and spinal cord were removed from anesthetized adult male Sprague-Dawley rats under a dissecting microscope. The tissues were frozen in dry ice and homogenized with a Polytron homogenizer; the RNA was extracted by the guanidine isothiocyanate procedure (Chomczynksi, et al., Anal. Biochemistry 162:156-159, (1987)). Total RNA (5 μg of each sample) was dissolved in MEN buffer containing 50% formamide, 6.6% formaldehyde and denatured at 65° C. for 5-10 minutes. The RNA was electrophoresed through a 0.8% agarose gel containing 8.3% formaldehyde in MEN buffer. The electrode buffer was MEN buffer containing 3.7% formaldehyde; the gel was run at 50 V for 12-18 hours.

After electrophoresis, the gel was rinsed in 2×SSC and the RNA was transferred to a Duralose membrane (Stratagene) with 20×SSC by capillary action; the membrane was baked under vacuum at 80° C. for 1 hour. The membrane was prehybridized in 50% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.1, 1× Denhardt's solution, 0.5% SDS, and sheared, heat-denatured salmon sperm DNA (1 mg/ml) for 16 hours at 42° C. The membrane was hybridized in 50% formamide, 5×SSC, 50 mM sodium phosphate, pH 7.1, 1× Denhardt's solution, 0.5% SDS, and sheared, heat-denatured salmon sperm DNA (200 μg/ml) with a ³²P-labeled cRNA probe (ca. 1-3×10⁶ cpm/ml). The probe was the cloned fragment, CNaD4-2, which contains the Domain 4 sequence of PN4 sodium channel α-subunit sequence. The probe was hybridized for 18 hours at 42° C. The cRNA probe was synthesized by excising and subcloning the fragment into pBluescript KS+ vector, purchased from Stratagene. The cRNA was transcribed in vitro using T3 RNA polymerase, purchased from Promega, after linearizing the plasmid with Xbal, purchased from Boehringer Mannheim. Protocols for each procedure mentioned above can be found in Molecular Cloning: A Laboratory Manual by Sambrook, et al. (Cold Spring Harbor Laboratory Press, 2nd edition, 1989).

The membrane was washed three times with 2×SSC, 0.1% SDS at room temperature for 20 minutes and then washed once with 0.1×SSC, 0.1% SDS at 68° C. for 30 minutes. The filter was exposed against Kodak X-omat AR film at −80° C. with intensifying screens for up to two weeks.

Size markers, including ribosomal 18S and 28S RNAs and RNA markers (GIBCO BRL), were run in parallel lanes of the gel. Their positions were determined by staining the excised lane with ethidium bromide (0.5 μg/ml) followed by photography under UV light. The CNaD4-2 probe hybridized to RNA from the brain, cerebellum, dorsal, and ventral horn of the spinal cord with sizes of 11 kb, 9.5 kb, 7.5 kb, and 6.5 kb, estimated on the basis of their positions relative to the standards.

Bands of the same size were detected in a blot containing total RNA from DRG from neuropathic pain model. However, no signal was detected with RNA from naive DRG.

PN4 constitutes a subfamily of novel sodium channel genes; these genes are different from those detectable with other probes (e.g., PEAF8 and PN3 probes), as discussed in copending application no. 08/511,828. Sequence comparison of PN4 with NaCh6 (MRNA size=9.5kb) (Schaller, et al., J. Neurosci. 15:3231-3242 (1995)), Scn8a (Burgess, et al., Nature Genetics 10:461465 (1995)), and cardiac-specific sodium channel for which only a partial sequence is available (mRNA size=7kb) (Sills, et al., J. Clin. Invest. 84:331-336 (1989)) indicates that these genes share a higher homology among themselves than with members of other sodium channel subfamilies such as the brain-type sodium channels, the TTX-insensitive cardiac sodium channel, and the TTX-resistant PN3 (copending application no. 08/511,828).

Semiquantitation of the signal intensity of the various bands detected in the blot containing RNAs from the neuropathic pain model indicated that the level cf 7.5kb transcript was upregulated ˜35 fold as compared with the DRG from the sham operated side on day 1 after the surgery, wherein the sciatic nerve was ligated with four loose ligatures causing a constriction injury. None of the other transcripts detected by the CNaD4-2 probe was regulated so dramatically. By day 2, the regulation was reduced to ˜5 fold as compared with the sham operated side. The experiment was performed with DRG pooled from 6 rats. This experimental data suggests that PN4, or its splice variant, PN4a, is involved in the pathophysiology of neuropathic pain.

EXAMPLE 7

Expression of full length PN4 and PN4a clones in Xenopur oocytes

After linearization with NotI, cRNA was prepared from pBSTAcIIr_PN4, pBSTAcIr_PN4a, and constructs of rat β₁ and rat β₂ in pBSTA, using a T7 in vitro transcription kit (mMessage mMachine, Ambion), and was injected into stage V and VI Xenopur oocytes using a Nanojector (Drummond), as described in Goldin, supra. After 1.5 days at 20° C., the oocytes were impaled with agarose-cushion electrodes (0.3-0.8 MOhm) and voltage-clamped with a Geneclamp 500 amplifier (Axon Instruments) in TEV mode; see Schreibmayer, et al., Pflugers Arch. 426:453-458 (1994). Stimulation and recording were controlled by a computer running pClamp (Axon Instruments), Kegel, et al., J. Neurosci. Meth. 12:317-330 (1982). Oocytes were perfused with a solution containing: 81 mM NaCl, 2 mM KC1, 1 mM MgCl₂, 0.3 mM CaCl₂, 20 mM Hepes-NaOH, pH 7.5. The data collected is shown in the FIGS. 7-9 and is described hereinafter.

FIG. 7a shows the currents produced from a PN4 sodium channel expressed in aXenopus oocyte using P/−4 leak subtraction (Benzanilla and Armstrong J. Gen. Physiol. 70:549-566 (1977)), filtered at 5 kHz with a 4-pole Bessel filter, and sampled at 50 kHz. The x-axis denotes time in milliseconds.

FIG. 7b illustrates the voltage to current relationship of the PN4 sodium channel expressed in a Xenopus oocyte. In the expression of PN4, 0.2 ng of cRNA gave 1.4±0.19 μA (n=9). In the expression of PN4a, 0.1 ng gave 1.8±0.23 μA (n=6).

FIGS. 8a and b show steady state inactivation of sodium currents produced by PN4 in Xenopur oocytes , using 10 second conditioning prepulses. In FIG. 8a, the x-axis denotes time in milliseconds. Leak currents were measured during long pulses to −100 mV and −120 mV, and the test currents corrected assuming that the leak currents had a linear current-voltage relationship. In FIG. 8b, the x-axis is conditioning potential in millivolts and the y-axis is current in μA. For the steady state inactivation of PN4, we found V_(½)=−70.7±0.71 mV, k=5.5±0.55 mV (n=3) For the steady state inactivation of PN4a, we found V_(½)=−73.3±0.97 mV, k=5.5±0.28 mV (n=4). Under these conditions, a V_(½) for inactivation of about −70 mV is similar to most sodium channels.

FIG. 9 demonstrates the effects of the β₁ and βsubunits upon PN4 function in Xenopus oocytes. Shown are currents produced when the PN4 α subunit is injected (a) alone; (b) with the β₁ subunit; (c) with the β₂ subunit; and (d) with both the β₁ and β₂ subunits. The x-axis in each of these figures denotes time in milliseconds. As these figures show, the inactivation kinetics of functionally active PN4 channels are accelerated by the β₁ subunit. No obvious effects are seen with the β₂ subunit.

As is seen in FIGS. 7a and 7 b, expression of PN4 produced an inward current with slow inactivation kinetics, similar to that of the rBIIa (Patton, et al., Neuron 7:637-647 (1991)) and rSkM1 α-subunits expressed in the absence of the β₁-subunit. Co-injection of rat β₁ cRNA (1 ng/oocyte) with PN4 cRNA accelerated inactivation kinetics of the channel, as seen in FIGS. 9a and 9b. Acceleration of inactivation kinetics of rBIIa and rSkml expressed in oocytes by co-expression of rat PI has been reported (Isom, et al., Science 256:839-842 (1992); and Waliner, et al., FEBS Lett. 336:535-539 1993)).

Sodium channels are distinctively sensitive or insensitive to neurotoxins such as TTX. The TTX-sensitive brain and skeletal muscle sodium channels are blocked by nanomolar TTX concentrations, whereas the TTX-insensitive cardiac sodium channels are blocked by micromolar TTX concentrations. In rat heart sodium channel 1, Cys³⁷⁴ is a critical determinant of TTX-insensitivity, as shown in Satin, et al., Science 256:1202-1205(1992); in the TTX-sensitive rBI, rBII, rBIII, and rSkM1, the corresponding residue is either Phe or Tyr. In PN4 and PN4a, this residue is Tyr. When expressed in Xenopur oocytes , we found the PN4 sodium current to be inhibited in a concentration-dependent manner by 0.1-10 nM TTX, with IC₅₀ values of 0.4 nM and 1.6 nM in two oocytes.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

43 5977 base pairs nucleic acid single linear DNA (genomic) 1 CTCGAGCCCG GGCAAGAGAA GATGGCAGCG CGGCTGCTCG CACCACCAGG CCCTGATAGT 60 TTCAAGCCTT TCACCCCTGA GTCGCTGGCA AACATCGAGA GGCGTATTGC CGAGAGCAAG 120 CTCAAGAAAC CACCAAAGGC GGATGGCAGC CACCGGGAGG ACGATGAAGA CAGCAAGCCC 180 AAGCCAAACA GTGACCTGGA GGCTGGGAAG AGTTTGCCTT TCATCTACGG GGACATCCCG 240 CAAGGCCTGG TTGCGGTTCC CCTGGAGGAC TTTGACCCTT ACTATTTGAC GCAGAAAACC 300 TTTGTAGTAT TAAACAGAGG GAAAACTCTC TTCAGATTTA GTGCCACACC TGCCTTGTAC 360 ATTTTAAGCC CTTTTAACCT GATAAGAAGA ATAGCTATTA AAATTTTGAT ACACTCAGTT 420 TTCAGCATGA TCATCATGTG CACCATCCTG ACCAACTGTG TGTTCATGAC CTTTAGTAAC 480 CCTCCAGAAT GGTCCAAGAA TGTGGAGTAC ACATTCACAG GGATTTACAC ATTTGAATCA 540 CTAGTGAAAA TCATCGCAAG AGGTTTCTGC ATAGACGGCT TCACCTTCTT GCGAGACCCG 600 TGGAACTGGT TAGACTTCAG TGTCATCATG ATGGCATATG TGACAGAGTT TGTGGACCTG 660 GGCAATGTCT CAGCGCTGAG AACATTCAGG GTTCTCCGAG CTTTGAAAAC TATCTCTGTA 720 ATTCCAGGCC TGAAGACAAT CGTGGGCGCC CTAATCCAGT CCGTGAAGAA GCTGTCGGAC 780 GTGATGATCC TGACAGTGTT CTGCCTGAGT GTTTTCGCCC TGATTGGCCT GCAGCTCTTC 840 ATGGGGAACC TTCGAAACAA GTGTGTCGTG TGGCCCATAA ACTTCAACGA GAGCTACCTG 900 GAGAACGGCA CCAGAGGCTT TGACTGGGAG GAATATATCA ACAATAAAAC AAACTTTTAC 960 ATGGTTCCTG GCATGCTAGA ACCCTTGCTC TGCGGGAACA GTTCTGATGC TGGGCAATGC 1020 CCAGAGGGAT TCCAGTGCAT GAAAGCAGGA AGGAACCCCA ACTACGGTTA CACCAGCTTT 1080 GACACCTTCA GCTGGGCCTT CTTGGCATTA TTCCGCCTTA TGACCCAGGA CTATTGGGAG 1140 AACTTATACC AGCTGACCTT ACGAGCCGCT GGGAAAACGT ACATGATCTT CTTTGTCTTG 1200 GTCATCTTCG TGGGTTCTTT CTATCTGGTG AACTTGATCT TGGCTGTGGT GGCCATGGCT 1260 TATGAGGAAC AGAACCAGGC AACACTGGAG GAGGCAGAGC AAAAAGAGGC CGAGTTCAAG 1320 GCAATGCTGG AGCAACTCAA GAAGCAGCAG GAGGAGGCAC AGGCTGCTGC AATGGCCACC 1380 TCAGCGGGCA CTGTCTCGGA AGACGCCATT GAAGAAGAAG GGGAAGATGG GGTAGGCTCT 1440 CCGAGGAGCT CTTCTGAACT GTCTAAACTC AGTTCCAAGA GCGCGAAGGA GCGGCGGAAC 1500 CGACGGAAGA AGAGGAAGCA GAAGGAGCTC TCTGAAGGCG AGGAGAAAGG GGACCCGGAG 1560 AAGGTGTTTA AGTCAGAGTC GGAAGACGGT ATGAGAAGGA AGGCCTTCCG GCTGCCAGAC 1620 AACAGGATAG GGAGGAAGTT TTCCATCATG AATCAGTCGC TGCTCAGCAT TCCAGGCTCG 1680 CCCTTCCTCT CCCGACATAA CAGCAAAAGC AGCATCTTCA GCTTCCGGGG ACCCGGTCGG 1740 TTCCGGGACC CCGGCTCTGA GAATGAGTTC GCAGACGATG AACACAGCAC CGTGGAGGAG 1800 AGCGAGGGCC GGCGTGACTC GCTCTTCATC CCGATCCGCG CCCGCGAGCG CCGCAGCAGC 1860 TACAGTGGCT ACAGCGGCTA CAGCCAGTGC AGCCGCTCGT CGCGCATCTT CCCCAGCCTG 1920 CGGCGCAGCG TGAAGCGCAA CAGCACGGTG GACTGCAACG GCGTAGTGTC ACTCATCGGG 1980 CCCGGCTCAC ACATCGGGCG GCTCCTGCCT GAGGCAACGA CTGAGGTGGA AATTAAGAAG 2040 AAAGGCCCTG GATCTCTTTT AGTTTCTATG GACCAACTCG CCTCCTACGG ACGGAAGGAC 2100 AGAATCAACA GCATAATGAG CGTGGTCACA AACACGCTAG TGGAAGAGCT GGAAGAGTCT 2160 CAGAGAAAGT GCCCACCGTG CTGGTATAAG TTTGCCAACA CTTTCCTCAT CTGGGAGTGT 2220 CACCCCTACT GGATAAAACT GAAGGAGATC GTGAACTTAA TCGTCATGGA CCCTTTTGTA 2280 GACTTAGCCA TCACCATCTG CATCGTTCTG AATACGCTAT TTATGGCAAT GGAGCACCAT 2340 CCCATGACAC CACAGTTCGA ACACGTCTTG GCCGTAGGAA ATCTGGTGTT CACCGGGATC 2400 TTCACGGCGG AAATGTTTCT GAAGCTCATA GCCATGGACC CCTACTATTA TTTCCAAGAA 2460 GGCTGGAACA TTTTTGACGG ATTTATTGTC TCCCTCAGTT TAATGGAGCT GAGTCTCGCA 2520 GATGTGGAGG GGCTCTCAGT GCTGCGGTCT TTCCGACTGC TCCGAGTCTT CAAGCTGGCC 2580 AAGTCCTGGC CCACCCTGAA CATGCTGATC AAGATCATCG GGAACTCCGT GGGTGCCCTG 2640 GGCAACCTGA CCCTGGTGCT GGCCATCATC GTCTTCATCT TCGCCGTGGT GGGGATGCAG 2700 CTGTTTGGAA AGAGTTACAA GGAGTGCGTC TGTAAGATCA ACCAGGAGTG CAAGCTCCCG 2760 CGCTGGCACA TGAACGACTT CTTCCACTCC TTCCTCATCG TCTTCCGAGT GCTGTGTGGG 2820 GAGTGGATCG AGACCATGTG GGACTGCATG GAGGTGGCCG GCCAGGCCAT GTGCCTCATT 2880 GTCTTCATGA TGGTTATGGT CATTGGCAAC CTGGTGGTGC TGAATCTATT CCTGGCCTTG 2940 CTTCTGAGCT CCTTCAGCGC AGACAACCTG GCGGCCACAG ACGACGACGG GGAAATGAAC 3000 AACCTGCAGA TCTCAGTGAT CCGGATCAAG AAGGGCGTGG CCTGGACCAA AGTGAAGGTG 3060 CACGCCTTCA TGCAGGCTCA CTTCAAGCAG CGGGAGGCGG ATGAAGTGAA ACCCCTCGAC 3120 GAGCTGTATG AGAAGAAGGC CAACTGCATC GCCAACCACA CGGGCGTGGA TATCCACCGG 3180 AACGGCGACT TCCAGAAGAA CGGGAACGGA ACCACCAGCG GCATCGGCAG CAGCGTGGAG 3240 AAGTACATCA TCGACGAGGA CCACATGTCC TTCATTAACA ACCCAAACCT GACCGTCCGG 3300 GTGCCCATTG CTGTGGGCGA GTCTGACTTC GAGAACCTCA ACACAGAGGA TGTTAGCAGC 3360 GAATCAGACC CTGAAGGCAG CAAAGATAAA CTGGACGATA CCAGCTCCTC AGAAGGAAGT 3420 ACCATCGACA TCAAGCCTGA GGTGGAAGAA GTTCCCGTGG AGCAACCTGA GGAATACTTG 3480 GATCCGGACG CCTGCTTTAC AGAGGGTTGC GTCCAGCGGT TCAAGTGCTG CCAGGTCAAC 3540 ATCGAGGAAG GACTAGGCAA GTCGTGGTGG ATCTTGCGGA AAACCTGCTT CCTCATTGTG 3600 GAGCACAATT GGTTTGAGAC CTTCATCATC TTCATGATTC TGCTCAGCAG TGGCGCCCTG 3660 GCCTTTGAGG ACATCTACAT TGAGCAGAGG AAGACCATCC GCACCATCCT GGAGTATGCG 3720 GACAAGGTCT TCACCTACAT CTTCATCCTG GAGATGTTGC TCAAGTGGAC AGCCTACGGC 3780 TTCGTCAAGT TCTTCACCAA TGCCTGGTGC TGGTTGGACT TCCTCATTGT GGCTGTCTCT 3840 TTAGTCAGCC TTATAGCTAA TGCCCTGGGC TACTCGGAAC TAGGTGCCAT AAAGTCCCTT 3900 AGGACCCTAA GAGCTTTGAG ACCCTTAAGA GCCTTATCAC GATTTGAAGG GATGAGGGTG 3960 GTGGTGAATG CCTTGGTGGG CGCCATCCCC TCCATCATGA ATGTGCTGCT GGTGTGTCTC 4020 ATCTTCTGGC TGATTTTCAG CATCATGGGA GTTAACCTGT TTGCGGGGAA ATACCACTAC 4080 TGCTTTAATG AGACTTCTGA AATCCGGTTC GAAATCGATA TTGTCAACAA TAAAACGGAC 4140 TGTGAGAAGC TCATGGAGGG CAACAGCACG GAGATCCGAT GGAAGAATGT CAAGATCAAC 4200 TTTGACAATG TCGGAGCAGG GTACCTGGCC CTTCTTCAAG TGGCAACCTT CAAAGGCTGG 4260 ATGGACATCA TGTATGCGGC TGTAGATTCC CGAAAGCCAG ACGAGCAGCC TGACTACGAG 4320 GGCAACATCT ACATGTACAT CTACTTCGTC ATCTTCATCA TCTTCGGCTC CTTCTTCACC 4380 CTCAACCTGT TCATCGGTGT CATCATCGAC AACTTCAACC AGCAGAAGAA AAAGTTTGGA 4440 GGTCAGGACA TCTTCATGAC AGAGGAACAG AAGAAGTACT ACAATGCCAT GAAAAAGCTG 4500 GGCTCCAAGA AGCCACAGAA GCCCATCCCC CGACCCTTGA ACAAAATCCA AGGGATTGTC 4560 TTTGATTTCG TCACTCAACA AGCCTTTGAC ATTGTGATCA TGATGCTCAT CTGCCTTAAC 4620 ATGGTGACAA TGATGGTGGA GACAGACACT CAGAGCAAGC AGATGGAGAA CATTCTTTAC 4680 TGGATTAATC TGGTCTTTGT CATCTTCTTC ACCTGCGAGT GTGTGCTCAA AATGTTTGCC 4740 TTGAGACACT ACTATTTCAC CATTGGCTGG AACATCTTTG ACTTTGTGGT GGTCATCCTC 4800 TCCATTGTGG GAATGTTCCT GGCTGATATC ATTGAGAAGT ACTTCGTCTC CCCAACCCTA 4860 TTCCGAGTTA TCCGATTGGC CCGTATTGGG CGCATCTTGC GTCTGATCAA GGGCGCCAAA 4920 GGGATCCGCA CCCTGCTCTT TGCCTTAATG ATGTCGCTGC CCGCCCTGTT CAACATCGGC 4980 CTCCTGCTCT TCCTCGTCAT GTTCATCTTC TCCATTTTTG GCATGTCCAA CTTCGCATAC 5040 GTGAAGCACG AGGCCGGCAT TGACGACATG TTCAACTTCG AGACATTTGG CAACAGCATG 5100 ATCTGTTTGT TCCAGATCAC AACGTCTGCT GGCTGGGATG GCCTGCTGCT GCCAATCCTG 5160 AACCGCCCCC CTGACTGCAG CTTGGACAAA GAGCACCCAG GGAGTGGCTT CAAAGGGGAC 5220 TGTGGGAACC CCTCGGTGGG CATCTTCTTC TTTGTGAGCT ACATCATCAT CTCCTTCCTG 5280 ATTGTGGTGA ACATGTACAT CGCCATCATC CTGGAGAACT TCAGCGTGGC CACCGAGGAG 5340 AGCGCCGACC CTCTGAGTGA GGATGACTTC GAGACTTTCT ATGAGATCTG GGAGAAGTTT 5400 GACCCAGACG CCACCCAGTT CATCGAGTAC TGTAAGCTGG CAGACTTTGC CGACGCCCTG 5460 GAGCACCCGC TCCGAGTACC CAAGCCCAAC ACCATCGAGC TCATCGCCAT GGACCTGCCC 5520 ATGGTGAGCG GAGATCGCAT CCACTGCTTG GACATCCTTT TCGCCTTCAC CAAGCGAGTC 5580 CTGGGAGACA GTGGGGAGTT GGACATCCTG CGGCAGCAGA TGGAGGAGCG GTTCGTGGCA 5640 TCCAATCCTT CCAAAGTGTC TTACGAGCCT ATCACAACCA CTCTGCGGCG CAAGCAGGAG 5700 GAGGTGTCTG CAGTGGTCCT GCAGCGTGCC TACAGGGGAC ACTTGGCTAG GCGGGGCTTC 5760 ATCTGCAGAA AGATGGCCTC CAACAAGCTG GAGAATGGAG GCACACACAG AGACAAGAAG 5820 GAGAGCACCC CGTCCACAGC CTCCCTCCCC TCTTACGACA GCGTCACAAA GCCAGACAAG 5880 GAGAAGCAGC AGCGTGCGGA GGAGGGCAGA AGGGAAAGAG CCAAGAGGCA AAAAGAGGTC 5940 AGGGAGTCCA AGTGCTAGAG GAGGGGAAAG GAAGCTT 5977 6007 base pairs nucleic acid single linear DNA (genomic) 2 CTCGAGCCCG GGCAAGAGAA GATGGCAGCG CGGCTGCTCG CACCACCAGG CCCTGATAGT 60 TTCAAGCCTT TCACCCCTGA GTCGCTGGCA AACATCGAGA GGCGTATTGC CGAGAGCAAG 120 CTCAAGAAAC CACCAAAGGC GGATGGCAGC CACCGGGAGG ACGATGAAGA CAGCAAGCCC 180 AAGCCAAACA GTGACCTGGA GGCTGGGAAG AGTTTGCCTT TCATCTACGG GGACATCCCG 240 CAAGGCCTGG TTGCGGTTCC CCTGGAGGAC TTTGACCCTT ACTATTTGAC GCAGAAAACC 300 TTTGTAGTAT TAAACAGAGG GAAAACTCTC TTCAGATTTA GTGCCACACC TGCCTTGTAC 360 ATTTTAAGCC CTTTTAACCT GATAAGAAGA ATAGCTATTA AAATTTTGAT ACACTCAGTT 420 TTCAGCATGA TCATCATGTG CACCATCCTG ACCAACTGTG TGTTCATGAC CTTTAGTAAC 480 CCTCCAGAAT GGTCCAAGAA TGTGGAGTAC ACATTCACAG GGATTTACAC ATTTGAATCA 540 CTAGTGAAAA TCATCGCAAG AGGTTTCTGC ATAGACGGCT TCACCTTCTT GCGAGACCCG 600 TGGAACTGGT TAGACTTCAG TGTCATCATG ATGGCATATG TGACAGAGTT TGTGGACCTG 660 GGCAATGTCT CAGCGCTGAG AACATTCAGG GTTCTCCGAG CTTTGAAAAC TATCTCTGTA 720 ATTCCAGGCC TGAAGACAAT CGTGGGCGCC CTAATCCAGT CCGTGAAGAA GCTGTCGGAC 780 GTGATGATCC TGACAGTGTT CTGCCTGAGT GTTTTCGCCC TGATTGGCCT GCAGCTCTTC 840 ATGGGGAACC TTCGAAACAA GTGTGTCGTG TGGCCCATAA ACTTCAACGA GAGCTACCTG 900 GAGAACGGCA CCAGAGGCTT TGACTGGGAG GAATATATCA ACAATAAAAC AAACTTTTAC 960 ATGGTTCCTG GCATGCTAGA ACCCTTGCTC TGCGGGAACA GTTCTGATGC TGGGCAATGC 1020 CCAGAGGGAT TCCAGTGCAT GAAAGCAGGA AGGAACCCCA ACTACGGTTA CACCAGCTTT 1080 GACACCTTCA GCTGGGCCTT CTTGGCATTA TTCCGCCTTA TGACCCAGGA CTATTGGGAG 1140 AACTTATACC AGCTGACCTT ACGAGCCGCT GGGAAAACGT ACATGATCTT CTTTGTCTTG 1200 GTCATCTTCG TGGGTTCTTT CTATCTGGTG AACTTGATCT TGGCTGTGGT GGCCATGGCT 1260 TATGAGGAAC AGAACCAGGC AACACTGGAG GAGGCAGAGC AAAAAGAGGC CGAGTTCAAG 1320 GCAATGCTGG AGCAACTCAA GAAGCAGCAG GAGGAGGCAC AGGCTGCTGC AATGGCCACC 1380 TCAGCGGGCA CTGTCTCGGA AGACGCCATT GAAGAAGAAG GGGAAGATGG GGTAGGCTCT 1440 CCGAGGAGCT CTTCTGAACT GTCTAAACTC AGTTCCAAGA GCGCGAAGGA GCGGCGGAAC 1500 CGACGGAAGA AGAGGAAGCA GAAGGAGCTC TCTGAAGGCG AGGAGAAAGG GGACCCGGAG 1560 AAGGTGTTTA AGTCAGAGTC GGAAGACGGT ATGAGAAGGA AGGCCTTCCG GCTGCCAGAC 1620 AACAGGATAG GGAGGAAGTT TTCCATCATG AATCAGTCGC TGCTCAGCAT TCCAGGCTCG 1680 CCCTTCCTCT CCCGACATAA CAGCAAAAGC AGCATCTTCA GCTTCCGGGG ACCCGGTCGG 1740 TTCCGGGACC CCGGCTCTGA GAATGAGTTC GCAGACGATG AACACAGCAC CGTGGAGGAG 1800 AGCGAGGGCC GGCGTGACTC GCTCTTCATC CCGATCCGCG CCCGCGAGCG CCGCAGCAGC 1860 TACAGTGGCT ACAGCGGCTA CAGCCAGTGC AGCCGCTCGT CGCGCATCTT CCCCAGCCTG 1920 CGGCGCAGCG TGAAGCGCAA CAGCACGGTG GACTGCAACG GCGTAGTGTC ACTCATCGGG 1980 CCCGGCTCAC ACATCGGGCG GCTCCTGCCT GAGGTGAAAA TAGATAAGGC AGCTACGGAC 2040 AGCGCAACGA CTGAGGTGGA AATTAAGAAG AAAGGCCCTG GATCTCTTTT AGTTTCTATG 2100 GACCAACTCG CCTCCTACGG ACGGAAGGAC AGAATCAACA GCATAATGAG CGTGGTCACA 2160 AACACGCTAG TGGAAGAGCT GGAAGAGTCT CAGAGAAAGT GCCCACCGTG CTGGTATAAG 2220 TTTGCCAACA CTTTCCTCAT CTGGGAGTGT CACCCCTACT GGATAAAACT GAAGGAGATC 2280 GTGAACTTAA TCGTCATGGA CCCTTTTGTA GACTTAGCCA TCACCATCTG CATCGTTCTG 2340 AATACGCTAT TTATGGCAAT GGAGCACCAT CCCATGACAC CACAGTTCGA ACACGTCTTG 2400 GCCGTAGGAA ATCTGGTGTT CACCGGGATC TTCACGGCGG AAATGTTTCT GAAGCTCATA 2460 GCCATGGACC CCTACTATTA TTTCCAAGAA GGCTGGAACA TTTTTGACGG ATTTATTGTC 2520 TCCCTCAGTT TAATGGAGCT GAGTCTCGCA GATGTGGAGG GGCTCTCAGT GCTGCGGTCT 2580 TTCCGACTGC TCCGAGTCTT CAAGCTGGCC AAGTCCTGGC CCACCCTGAA CATGCTGATC 2640 AAGATCATCG GGAACTCCGT GGGTGCCCTG GGCAACCTGA CCCTGGTGCT GGCCATCATC 2700 GTCTTCATCT TCGCCGTGGT GGGGATGCAG CTGTTTGGAA AGAGTTACAA GGAGTGCGTC 2760 TGTAAGATCA ACCAGGAGTG CAAGCTCCCG CGCTGGCACA TGAACGACTT CTTCCACTCC 2820 TTCCTCATCG TCTTCCGAGT GCTGTGTGGG GAGTGGATCG AGACCATGTG GGACTGCATG 2880 GAGGTGGCCG GCCAGGCCAT GTGCCTCATT GTCTTCATGA TGGTTATGGT CATTGGCAAC 2940 CTGGTGGTGC TGAATCTATT CCTGGCCTTG CTTCTGAGCT CCTTCAGCGC AGACAACCTG 3000 GCGGCCACAG ACGACGACGG GGAAATGAAC AACCTGCAGA TCTCAGTGAT CCGGATCAAG 3060 AAGGGCGTGG CCTGGACCAA AGTGAAGGTG CACGCCTTCA TGCAGGCTCA CTTCAAGCAG 3120 CGGGAGGCGG ATGAAGTGAA ACCCCTCGAC GAGCTGTATG AGAAGAAGGC CAACTGCATC 3180 GCCAACCACA CGGGCGTGGA TATCCACCGG AACGGCGACT TCCAGAAGAA CGGGAACGGA 3240 ACCACCAGCG GCATCGGCAG CAGCGTGGAG AAGTACATCA TCGACGAGGA CCACATGTCC 3300 TTCATTAACA ACCCAAACCT GACCGTCCGG GTGCCCATTG CTGTGGGCGA GTCTGACTTC 3360 GAGAACCTCA ACACAGAGGA TGTTAGCAGC GAATCAGACC CTGAAGGCAG CAAAGATAAA 3420 CTGGACGATA CCAGCTCCTC AGAAGGAAGT ACCATCGACA TCAAGCCTGA GGTGGAAGAA 3480 GTTCCCGTGG AGCAACCTGA GGAATACTTG GATCCGGACG CCTGCTTTAC AGAGGGTTGC 3540 GTCCAGCGGT TCAAGTGCTG CCAGGTCAAC ATCGAGGAAG GACTAGGCAA GTCGTGGTGG 3600 ATCTTGCGGA AAACCTGCTT CCTCATTGTG GAGCACAATT GGTTTGAGAC CTTCATCATC 3660 TTCATGATTC TGCTCAGCAG TGGCGCCCTG GCCTTTGAGG ACATCTACAT TGAGCAGAGG 3720 AAGACCATCC GCACCATCCT GGAGTATGCG GACAAGGTCT TCACCTACAT CTTCATCCTG 3780 GAGATGTTGC TCAAGTGGAC AGCCTACGGC TTCGTCAAGT TCTTCACCAA TGCCTGGTGC 3840 TGGTTGGACT TCCTCATTGT GGCTGTCTCT TTAGTCAGCC TTATAGCTAA TGCCCTGGGC 3900 TACTCGGAAC TAGGTGCCAT AAAGTCCCTT AGGACCCTAA GAGCTTTGAG ACCCTTAAGA 3960 GCCTTATCAC GATTTGAAGG GATGAGGGTG GTGGTGAATG CCTTGGTGGG CGCCATCCCC 4020 TCCATCATGA ATGTGCTGCT GGTGTGTCTC ATCTTCTGGC TGATTTTCAG CATCATGGGA 4080 GTTAACCTGT TTGCGGGGAA ATACCACTAC TGCTTTAATG AGACTTCTGA AATCCGGTTC 4140 GAAATCGATA TTGTCAACAA TAAAACGGAC TGTGAGAAGC TCATGGAGGG CAACAGCACG 4200 GAGATCCGAT GGAAGAATGT CAAGATCAAC TTTGACAATG TCGGAGCAGG GTACCTGGCC 4260 CTTCTTCAAG TGGCAACCTT CAAAGGCTGG ATGGACATCA TGTATGCGGC TGTAGATTCC 4320 CGAAAGCCAG ACGAGCAGCC TGACTACGAG GGCAACATCT ACATGTACAT CTACTTCGTC 4380 ATCTTCATCA TCTTCGGCTC CTTCTTCACC CTCAACCTGT TCATCGGTGT CATCATCGAC 4440 AACTTCAACC AGCAGAAGAA AAAGTTTGGA GGTCAGGACA TCTTCATGAC AGAGGAACAG 4500 AAGAAGTACT ACAATGCCAT GAAAAAGCTG GGCTCCAAGA AGCCACAGAA GCCCATCCCC 4560 CGACCCTTGA ACAAAATCCA AGGGATTGTC TTTGATTTCG TCACTCAACA AGCCTTTGAC 4620 ATTGTGATCA TGATGCTCAT CTGCCTTAAC ATGGTGACAA TGATGGTGGA GACAGACACT 4680 CAGAGCAAGC AGATGGAGAA CATTCTTTAC TGGATTAATC TGGTCTTTGT CATCTTCTTC 4740 ACCTGCGAGT GTGTGCTCAA AATGTTTGCC TTGAGACACT ACTATTTCAC CATTGGCTGG 4800 AACATCTTTG ACTTTGTGGT GGTCATCCTC TCCATTGTGG GAATGTTCCT GGCTGATATC 4860 ATTGAGAAGT ACTTCGTCTC CCCAACCCTA TTCCGAGTTA TCCGATTGGC CCGTATTGGG 4920 CGCATCTTGC GTCTGATCAA GGGCGCCAAA GGGATCCGCA CCCTGCTCTT TGCCTTAATG 4980 ATGTCGCTGC CCGCCCTGTT CAACATCGGC CTCCTGCTCT TCCTCGTCAT GTTCATCTTC 5040 TCCATTTTTG GCATGTCCAA CTTCGCATAC GTGAAGCACG AGGCCGGCAT TGACGACATG 5100 TTCAACTTCG AGACATTTGG CAACAGCATG ATCTGTTTGT TCCAGATCAC AACGTCTGCT 5160 GGCTGGGATG GCCTGCTGCT GCCAATCCTG AACCGCCCCC CTGACTGCAG CTTGGACAAA 5220 GAGCACCCAG GGAGTGGCTT CAAAGGGGAC TGTGGGAACC CCTCGGTGGG CATCTTCTTC 5280 TTTGTGAGCT ACATCATCAT CTCCTTCCTG ATTGTGGTGA ACATGTACAT CGCCATCATC 5340 CTGGAGAACT TCAGCGTGGC CACCGAGGAG AGCGCCGACC CTCTGAGTGA GGATGACTTC 5400 GAGACTTTCT ATGAGATCTG GGAGAAGTTT GACCCAGACG CCACCCAGTT CATCGAGTAC 5460 TGTAAGCTGG CAGACTTTGC CGACGCCCTG GAGCACCCGC TCCGAGTACC CAAGCCCAAC 5520 ACCATCGAGC TCATCGCCAT GGACCTGCCC ATGGTGAGCG GAGATCGCAT CCACTGCTTG 5580 GACATCCTTT TCGCCTTCAC CAAGCGAGTC CTGGGAGACA GTGGGGAGTT GGACATCCTG 5640 CGGCAGCAGA TGGAGGAGCG GTTCGTGGCA TCCAATCCTT CCAAAGTGTC TTACGAGCCT 5700 ATCACAACCA CTCTGCGGCG CAAGCAGGAG GAGGTGTCTG CAGTGGTCCT GCAGCGTGCC 5760 TACAGGGGAC ACTTGGCTAG GCGGGGCTTC ATCTGCAGAA AGATGGCCTC CAACAAGCTG 5820 GAGAATGGAG GCACACACAG AGACAAGAAG GAGAGCACCC CGTCCACAGC CTCCCTCCCC 5880 TCTTACGACA GCGTCACAAA GCCAGACAAG GAGAAGCAGC AGCGTGCGGA GGAGGGCAGA 5940 AGGGAAAGAG CCAAGAGGCA AAAAGAGGTC AGGGAGTCCA AGTGCTAGAG GAGGGGAAAG 6000 GAAGCTT 6007 1978 amino acids amino acid single linear peptide 3 Met Ala Ala Arg Leu Leu Ala Pro Pro Gly Pro Asp Ser Phe Lys Pro 1 5 10 15 Phe Thr Pro Glu Ser Leu Ala Asn Ile Glu Arg Arg Ile Ala Glu Ser 20 25 30 Lys Leu Lys Lys Pro Pro Lys Ala Asp Gly Ser His Arg Glu Asp Asp 35 40 45 Glu Asp Ser Lys Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Ser 50 55 60 Leu Pro Phe Ile Tyr Gly Asp Ile Pro Gln Gly Leu Val Ala Val Pro 65 70 75 80 Leu Glu Asp Phe Asp Pro Tyr Tyr Leu Thr Gln Lys Thr Phe Val Val 85 90 95 Leu Asn Arg Gly Lys Thr Leu Phe Arg Phe Ser Ala Thr Pro Ala Leu 100 105 110 Tyr Ile Leu Ser Pro Phe Asn Leu Ile Arg Arg Ile Ala Ile Lys Ile 115 120 125 Leu Ile His Ser Val Phe Ser Met Ile Ile Met Cys Thr Ile Leu Thr 130 135 140 Asn Cys Val Phe Met Thr Phe Ser Asn Pro Pro Glu Trp Ser Lys Asn 145 150 155 160 Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys 165 170 175 Ile Ile Ala Arg Gly Phe Cys Ile Asp Gly Phe Thr Phe Leu Arg Asp 180 185 190 Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Met Met Ala Tyr Val Thr 195 200 205 Glu Phe Val Asp Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val 210 215 220 Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile 225 230 235 240 Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile 245 250 255 Leu Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu 260 265 270 Phe Met Gly Asn Leu Arg Asn Lys Cys Val Val Trp Pro Ile Asn Phe 275 280 285 Asn Glu Ser Tyr Leu Glu Asn Gly Thr Arg Gly Phe Asp Trp Glu Glu 290 295 300 Tyr Ile Asn Asn Lys Thr Asn Phe Tyr Met Val Pro Gly Met Leu Glu 305 310 315 320 Pro Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly 325 330 335 Phe Gln Cys Met Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser 340 345 350 Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met Thr 355 360 365 Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly 370 375 380 Lys Thr Tyr Met Ile Phe Phe Val Leu Val Ile Phe Val Gly Ser Phe 385 390 395 400 Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu 405 410 415 Gln Asn Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe 420 425 430 Lys Ala Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln Ala 435 440 445 Ala Ala Met Ala Thr Ser Ala Gly Thr Val Ser Glu Asp Ala Ile Glu 450 455 460 Glu Glu Gly Glu Asp Gly Val Gly Ser Pro Arg Ser Ser Ser Glu Leu 465 470 475 480 Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg Lys 485 490 495 Lys Arg Lys Gln Lys Glu Leu Ser Glu Gly Glu Glu Lys Gly Asp Pro 500 505 510 Glu Lys Val Phe Lys Ser Glu Ser Glu Asp Gly Met Arg Arg Lys Ala 515 520 525 Phe Arg Leu Pro Asp Asn Arg Ile Gly Arg Lys Phe Ser Ile Met Asn 530 535 540 Gln Ser Leu Leu Ser Ile Pro Gly Ser Pro Phe Leu Ser Arg His Asn 545 550 555 560 Ser Lys Ser Ser Ile Phe Ser Phe Arg Gly Pro Gly Arg Phe Arg Asp 565 570 575 Pro Gly Ser Glu Asn Glu Phe Ala Asp Asp Glu His Ser Thr Val Glu 580 585 590 Glu Ser Glu Gly Arg Arg Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg 595 600 605 Glu Arg Arg Ser Ser Tyr Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser 610 615 620 Arg Ser Ser Arg Ile Phe Pro Ser Leu Arg Arg Ser Val Lys Arg Asn 625 630 635 640 Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Ile Gly Pro Gly Ser 645 650 655 His Ile Gly Arg Leu Leu Pro Glu Ala Thr Thr Glu Val Glu Ile Lys 660 665 670 Lys Lys Gly Pro Gly Ser Leu Leu Val Ser Met Asp Gln Leu Ala Ser 675 680 685 Tyr Gly Arg Lys Asp Arg Ile Asn Ser Ile Met Ser Val Val Thr Asn 690 695 700 Thr Leu Val Glu Glu Leu Glu Glu Ser Gln Arg Lys Cys Pro Pro Cys 705 710 715 720 Trp Tyr Lys Phe Ala Asn Thr Phe Leu Ile Trp Glu Cys His Pro Tyr 725 730 735 Trp Ile Lys Leu Lys Glu Ile Val Asn Leu Ile Val Met Asp Pro Phe 740 745 750 Val Asp Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met 755 760 765 Ala Met Glu His His Pro Met Thr Pro Gln Phe Glu His Val Leu Ala 770 775 780 Val Gly Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Phe Leu 785 790 795 800 Lys Leu Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp Asn 805 810 815 Ile Phe Asp Gly Phe Ile Val Ser Leu Ser Leu Met Glu Leu Ser Leu 820 825 830 Ala Asp Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg 835 840 845 Val Phe Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys 850 855 860 Ile Ile Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu 865 870 875 880 Ala Ile Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly 885 890 895 Lys Ser Tyr Lys Glu Cys Val Cys Lys Ile Asn Gln Glu Cys Lys Leu 900 905 910 Pro Arg Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe 915 920 925 Arg Val Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu 930 935 940 Val Ala Gly Gln Ala Met Cys Leu Ile Val Phe Met Met Val Met Val 945 950 955 960 Ile Gly Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser 965 970 975 Ser Phe Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Gly Glu Met 980 985 990 Asn Asn Leu Gln Ile Ser Val Ile Arg Ile Lys Lys Gly Val Ala Trp 995 1000 1005 Thr Lys Val Lys Val His Ala Phe Met Gln Ala His Phe Lys Gln Arg 1010 1015 1020 Glu Ala Asp Glu Val Lys Pro Leu Asp Glu Leu Tyr Glu Lys Lys Ala 1025 1030 1035 1040 Asn Cys Ile Ala Asn His Thr Gly Val Asp Ile His Arg Asn Gly Asp 1045 1050 1055 Phe Gln Lys Asn Gly Asn Gly Thr Thr Ser Gly Ile Gly Ser Ser Val 1060 1065 1070 Glu Lys Tyr Ile Ile Asp Glu Asp His Met Ser Phe Ile Asn Asn Pro 1075 1080 1085 Asn Leu Thr Val Arg Val Pro Ile Ala Val Gly Glu Ser Asp Phe Glu 1090 1095 1100 Asn Leu Asn Thr Glu Asp Val Ser Ser Glu Ser Asp Pro Glu Gly Ser 1105 1110 1115 1120 Lys Asp Lys Leu Asp Asp Thr Ser Ser Ser Glu Gly Ser Thr Ile Asp 1125 1130 1135 Ile Lys Pro Glu Val Glu Glu Val Pro Val Glu Gln Pro Glu Glu Tyr 1140 1145 1150 Leu Asp Pro Asp Ala Cys Phe Thr Glu Gly Cys Val Gln Arg Phe Lys 1155 1160 1165 Cys Cys Gln Val Asn Ile Glu Glu Gly Leu Gly Lys Ser Trp Trp Ile 1170 1175 1180 Leu Arg Lys Thr Cys Phe Leu Ile Val Glu His Asn Trp Phe Glu Thr 1185 1190 1195 1200 Phe Ile Ile Phe Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu 1205 1210 1215 Asp Ile Tyr Ile Glu Gln Arg Lys Thr Ile Arg Thr Ile Leu Glu Tyr 1220 1225 1230 Ala Asp Lys Val Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys 1235 1240 1245 Trp Thr Ala Tyr Gly Phe Val Lys Phe Phe Thr Asn Ala Trp Cys Trp 1250 1255 1260 Leu Asp Phe Leu Ile Val Ala Val Ser Leu Val Ser Leu Ile Ala Asn 1265 1270 1275 1280 Ala Leu Gly Tyr Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu 1285 1290 1295 Arg Ala Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg 1300 1305 1310 Val Val Val Asn Ala Leu Val Gly Ala Ile Pro Ser Ile Met Asn Val 1315 1320 1325 Leu Leu Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val 1330 1335 1340 Asn Leu Phe Ala Gly Lys Tyr His Tyr Cys Phe Asn Glu Thr Ser Glu 1345 1350 1355 1360 Ile Arg Phe Glu Ile Asp Ile Val Asn Asn Lys Thr Asp Cys Glu Lys 1365 1370 1375 Leu Met Glu Gly Asn Ser Thr Glu Ile Arg Trp Lys Asn Val Lys Ile 1380 1385 1390 Asn Phe Asp Asn Val Gly Ala Gly Tyr Leu Ala Leu Leu Gln Val Ala 1395 1400 1405 Thr Phe Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Arg 1410 1415 1420 Lys Pro Asp Glu Gln Pro Asp Tyr Glu Gly Asn Ile Tyr Met Tyr Ile 1425 1430 1435 1440 Tyr Phe Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu 1445 1450 1455 Phe Ile Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Phe 1460 1465 1470 Gly Gly Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn 1475 1480 1485 Ala Met Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg 1490 1495 1500 Pro Leu Asn Lys Ile Gln Gly Ile Val Phe Asp Phe Val Thr Gln Gln 1505 1510 1515 1520 Ala Phe Asp Ile Val Ile Met Met Leu Ile Cys Leu Asn Met Val Thr 1525 1530 1535 Met Met Val Glu Thr Asp Thr Gln Ser Lys Gln Met Glu Asn Ile Leu 1540 1545 1550 Tyr Trp Ile Asn Leu Val Phe Val Ile Phe Phe Thr Cys Glu Cys Val 1555 1560 1565 Leu Lys Met Phe Ala Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn 1570 1575 1580 Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu 1585 1590 1595 1600 Ala Asp Ile Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val 1605 1610 1615 Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala 1620 1625 1630 Lys Gly Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala 1635 1640 1645 Leu Phe Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Phe Ser 1650 1655 1660 Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys His Glu Ala Gly Ile 1665 1670 1675 1680 Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu 1685 1690 1695 Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Leu Pro Ile 1700 1705 1710 Leu Asn Arg Pro Pro Asp Cys Ser Leu Asp Lys Glu His Pro Gly Ser 1715 1720 1725 Gly Phe Lys Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Phe 1730 1735 1740 Val Ser Tyr Ile Ile Ile Ser Phe Leu Ile Val Val Asn Met Tyr Ile 1745 1750 1755 1760 Ala Ile Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Ala Asp 1765 1770 1775 Pro Leu Ser Glu Asp Asp Phe Glu Thr Phe Tyr Glu Ile Trp Glu Lys 1780 1785 1790 Phe Asp Pro Asp Ala Thr Gln Phe Ile Glu Tyr Cys Lys Leu Ala Asp 1795 1800 1805 Phe Ala Asp Ala Leu Glu His Pro Leu Arg Val Pro Lys Pro Asn Thr 1810 1815 1820 Ile Glu Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile 1825 1830 1835 1840 His Cys Leu Asp Ile Leu Phe Ala Phe Thr Lys Arg Val Leu Gly Asp 1845 1850 1855 Ser Gly Glu Leu Asp Ile Leu Arg Gln Gln Met Glu Glu Arg Phe Val 1860 1865 1870 Ala Ser Asn Pro Ser Lys Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu 1875 1880 1885 Arg Arg Lys Gln Glu Glu Val Ser Ala Val Val Leu Gln Arg Ala Tyr 1890 1895 1900 Arg Gly His Leu Ala Arg Arg Gly Phe Ile Cys Arg Lys Met Ala Ser 1905 1910 1915 1920 Asn Lys Leu Glu Asn Gly Gly Thr His Arg Asp Lys Lys Glu Ser Thr 1925 1930 1935 Pro Ser Thr Ala Ser Leu Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp 1940 1945 1950 Lys Glu Lys Gln Gln Arg Ala Glu Glu Gly Arg Arg Glu Arg Ala Lys 1955 1960 1965 Arg Gln Lys Glu Val Arg Glu Ser Lys Cys 1970 1975 1988 amino acids amino acid single linear peptide 4 Met Ala Ala Arg Leu Leu Ala Pro Pro Gly Pro Asp Ser Phe Lys Pro 1 5 10 15 Phe Thr Pro Glu Ser Leu Ala Asn Ile Glu Arg Arg Ile Ala Glu Ser 20 25 30 Lys Leu Lys Lys Pro Pro Lys Ala Asp Gly Ser His Arg Glu Asp Asp 35 40 45 Glu Asp Ser Lys Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly Lys Ser 50 55 60 Leu Pro Phe Ile Tyr Gly Asp Ile Pro Gln Gly Leu Val Ala Val Pro 65 70 75 80 Leu Glu Asp Phe Asp Pro Tyr Tyr Leu Thr Gln Lys Thr Phe Val Val 85 90 95 Leu Asn Arg Gly Lys Thr Leu Phe Arg Phe Ser Ala Thr Pro Ala Leu 100 105 110 Tyr Ile Leu Ser Pro Phe Asn Leu Ile Arg Arg Ile Ala Ile Lys Ile 115 120 125 Leu Ile His Ser Val Phe Ser Met Ile Ile Met Cys Thr Ile Leu Thr 130 135 140 Asn Cys Val Phe Met Thr Phe Ser Asn Pro Pro Glu Trp Ser Lys Asn 145 150 155 160 Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu Val Lys 165 170 175 Ile Ile Ala Arg Gly Phe Cys Ile Asp Gly Phe Thr Phe Leu Arg Asp 180 185 190 Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Met Met Ala Tyr Val Thr 195 200 205 Glu Phe Val Asp Leu Gly Asn Val Ser Ala Leu Arg Thr Phe Arg Val 210 215 220 Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys Thr Ile 225 230 235 240 Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val Met Ile 245 250 255 Leu Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu Gln Leu 260 265 270 Phe Met Gly Asn Leu Arg Asn Lys Cys Val Val Trp Pro Ile Asn Phe 275 280 285 Asn Glu Ser Tyr Leu Glu Asn Gly Thr Arg Gly Phe Asp Trp Glu Glu 290 295 300 Tyr Ile Asn Asn Lys Thr Asn Phe Tyr Met Val Pro Gly Met Leu Glu 305 310 315 320 Pro Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Pro Glu Gly 325 330 335 Phe Gln Cys Met Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr Ser 340 345 350 Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met Thr 355 360 365 Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala Gly 370 375 380 Lys Thr Tyr Met Ile Phe Phe Val Leu Val Ile Phe Val Gly Ser Phe 385 390 395 400 Tyr Leu Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu Glu 405 410 415 Gln Asn Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu Phe 420 425 430 Lys Ala Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln Ala 435 440 445 Ala Ala Met Ala Thr Ser Ala Gly Thr Val Ser Glu Asp Ala Ile Glu 450 455 460 Glu Glu Gly Glu Asp Gly Val Gly Ser Pro Arg Ser Ser Ser Glu Leu 465 470 475 480 Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg Lys 485 490 495 Lys Arg Lys Gln Lys Glu Leu Ser Glu Gly Glu Glu Lys Gly Asp Pro 500 505 510 Glu Lys Val Phe Lys Ser Glu Ser Glu Asp Gly Met Arg Arg Lys Ala 515 520 525 Phe Arg Leu Pro Asp Asn Arg Ile Gly Arg Lys Phe Ser Ile Met Asn 530 535 540 Gln Ser Leu Leu Ser Ile Pro Gly Ser Pro Phe Leu Ser Arg His Asn 545 550 555 560 Ser Lys Ser Ser Ile Phe Ser Phe Arg Gly Pro Gly Arg Phe Arg Asp 565 570 575 Pro Gly Ser Glu Asn Glu Phe Ala Asp Asp Glu His Ser Thr Val Glu 580 585 590 Glu Ser Glu Gly Arg Arg Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg 595 600 605 Glu Arg Arg Ser Ser Tyr Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser 610 615 620 Arg Ser Ser Arg Ile Phe Pro Ser Leu Arg Arg Ser Val Lys Arg Asn 625 630 635 640 Ser Thr Val Asp Cys Asn Gly Val Val Ser Leu Ile Gly Pro Gly Ser 645 650 655 His Ile Gly Arg Leu Leu Pro Glu Val Lys Ile Asp Lys Ala Ala Thr 660 665 670 Asp Ser Ala Thr Thr Glu Val Glu Ile Lys Lys Lys Gly Pro Gly Ser 675 680 685 Leu Leu Val Ser Met Asp Gln Leu Ala Ser Tyr Gly Arg Lys Asp Arg 690 695 700 Ile Asn Ser Ile Met Ser Val Val Thr Asn Thr Leu Val Glu Glu Leu 705 710 715 720 Glu Glu Ser Gln Arg Lys Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn 725 730 735 Thr Phe Leu Ile Trp Glu Cys His Pro Tyr Trp Ile Lys Leu Lys Glu 740 745 750 Ile Val Asn Leu Ile Val Met Asp Pro Phe Val Asp Leu Ala Ile Thr 755 760 765 Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met Glu His His Pro 770 775 780 Met Thr Pro Gln Phe Glu His Val Leu Ala Val Gly Asn Leu Val Phe 785 790 795 800 Thr Gly Ile Phe Thr Ala Glu Met Phe Leu Lys Leu Ile Ala Met Asp 805 810 815 Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp Asn Ile Phe Asp Gly Phe Ile 820 825 830 Val Ser Leu Ser Leu Met Glu Leu Ser Leu Ala Asp Val Glu Gly Leu 835 840 845 Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe Lys Leu Ala Lys 850 855 860 Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile Gly Asn Ser Val 865 870 875 880 Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile Ile Val Phe Ile 885 890 895 Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser Tyr Lys Glu Cys 900 905 910 Val Cys Lys Ile Asn Gln Glu Cys Lys Leu Pro Arg Trp His Met Asn 915 920 925 Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg Val Leu Cys Gly Glu 930 935 940 Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val Ala Gly Gln Ala Met 945 950 955 960 Cys Leu Ile Val Phe Met Met Val Met Val Ile Gly Asn Leu Val Val 965 970 975 Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe Ser Ala Asp Asn 980 985 990 Leu Ala Ala Thr Asp Asp Asp Gly Glu Met Asn Asn Leu Gln Ile Ser 995 1000 1005 Val Ile Arg Ile Lys Lys Gly Val Ala Trp Thr Lys Val Lys Val His 1010 1015 1020 Ala Phe Met Gln Ala His Phe Lys Gln Arg Glu Ala Asp Glu Val Lys 1025 1030 1035 1040 Pro Leu Asp Glu Leu Tyr Glu Lys Lys Ala Asn Cys Ile Ala Asn His 1045 1050 1055 Thr Gly Val Asp Ile His Arg Asn Gly Asp Phe Gln Lys Asn Gly Asn 1060 1065 1070 Gly Thr Thr Ser Gly Ile Gly Ser Ser Val Glu Lys Tyr Ile Ile Asp 1075 1080 1085 Glu Asp His Met Ser Phe Ile Asn Asn Pro Asn Leu Thr Val Arg Val 1090 1095 1100 Pro Ile Ala Val Gly Glu Ser Asp Phe Glu Asn Leu Asn Thr Glu Asp 1105 1110 1115 1120 Val Ser Ser Glu Ser Asp Pro Glu Gly Ser Lys Asp Lys Leu Asp Asp 1125 1130 1135 Thr Ser Ser Ser Glu Gly Ser Thr Ile Asp Ile Lys Pro Glu Val Glu 1140 1145 1150 Glu Val Pro Val Glu Gln Pro Glu Glu Tyr Leu Asp Pro Asp Ala Cys 1155 1160 1165 Phe Thr Glu Gly Cys Val Gln Arg Phe Lys Cys Cys Gln Val Asn Ile 1170 1175 1180 Glu Glu Gly Leu Gly Lys Ser Trp Trp Ile Leu Arg Lys Thr Cys Phe 1185 1190 1195 1200 Leu Ile Val Glu His Asn Trp Phe Glu Thr Phe Ile Ile Phe Met Ile 1205 1210 1215 Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile Tyr Ile Glu Gln 1220 1225 1230 Arg Lys Thr Ile Arg Thr Ile Leu Glu Tyr Ala Asp Lys Val Phe Thr 1235 1240 1245 Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Thr Ala Tyr Gly Phe 1250 1255 1260 Val Lys Phe Phe Thr Asn Ala Trp Cys Trp Leu Asp Phe Leu Ile Val 1265 1270 1275 1280 Ala Val Ser Leu Val Ser Leu Ile Ala Asn Ala Leu Gly Tyr Ser Glu 1285 1290 1295 Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala Leu Arg Pro Leu 1300 1305 1310 Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val Val Asn Ala Leu 1315 1320 1325 Val Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu Val Cys Leu Ile 1330 1335 1340 Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu Phe Ala Gly Lys 1345 1350 1355 1360 Tyr His Tyr Cys Phe Asn Glu Thr Ser Glu Ile Arg Phe Glu Ile Asp 1365 1370 1375 Ile Val Asn Asn Lys Thr Asp Cys Glu Lys Leu Met Glu Gly Asn Ser 1380 1385 1390 Thr Glu Ile Arg Trp Lys Asn Val Lys Ile Asn Phe Asp Asn Val Gly 1395 1400 1405 Ala Gly Tyr Leu Ala Leu Leu Gln Val Ala Thr Phe Lys Gly Trp Met 1410 1415 1420 Asp Ile Met Tyr Ala Ala Val Asp Ser Arg Lys Pro Asp Glu Gln Pro 1425 1430 1435 1440 Asp Tyr Glu Gly Asn Ile Tyr Met Tyr Ile Tyr Phe Val Ile Phe Ile 1445 1450 1455 Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile Gly Val Ile Ile 1460 1465 1470 Asp Asn Phe Asn Gln Gln Lys Lys Lys Phe Gly Gly Gln Asp Ile Phe 1475 1480 1485 Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met Lys Lys Leu Gly 1490 1495 1500 Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Leu Asn Lys Ile Gln 1505 1510 1515 1520 Gly Ile Val Phe Asp Phe Val Thr Gln Gln Ala Phe Asp Ile Val Ile 1525 1530 1535 Met Met Leu Ile Cys Leu Asn Met Val Thr Met Met Val Glu Thr Asp 1540 1545 1550 Thr Gln Ser Lys Gln Met Glu Asn Ile Leu Tyr Trp Ile Asn Leu Val 1555 1560 1565 Phe Val Ile Phe Phe Thr Cys Glu Cys Val Leu Lys Met Phe Ala Leu 1570 1575 1580 Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe Asp Phe Val Val 1585 1590 1595 1600 Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Asp Ile Ile Glu Lys 1605 1610 1615 Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile 1620 1625 1630 Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr Leu 1635 1640 1645 Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe Asn Ile Gly Leu 1650 1655 1660 Leu Leu Phe Leu Val Met Phe Ile Phe Ser Ile Phe Gly Met Ser Asn 1665 1670 1675 1680 Phe Ala Tyr Val Lys His Glu Ala Gly Ile Asp Asp Met Phe Asn Phe 1685 1690 1695 Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser 1700 1705 1710 Ala Gly Trp Asp Gly Leu Leu Leu Pro Ile Leu Asn Arg Pro Pro Asp 1715 1720 1725 Cys Ser Leu Asp Lys Glu His Pro Gly Ser Gly Phe Lys Gly Asp Cys 1730 1735 1740 Gly Asn Pro Ser Val Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile 1745 1750 1755 1760 Ser Phe Leu Ile Val Val Asn Met Tyr Ile Ala Ile Ile Leu Glu Asn 1765 1770 1775 Phe Ser Val Ala Thr Glu Glu Ser Ala Asp Pro Leu Ser Glu Asp Asp 1780 1785 1790 Phe Glu Thr Phe Tyr Glu Ile Trp Glu Lys Phe Asp Pro Asp Ala Thr 1795 1800 1805 Gln Phe Ile Glu Tyr Cys Lys Leu Ala Asp Phe Ala Asp Ala Leu Glu 1810 1815 1820 His Pro Leu Arg Val Pro Lys Pro Asn Thr Ile Glu Leu Ile Ala Met 1825 1830 1835 1840 Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys Leu Asp Ile Leu 1845 1850 1855 Phe Ala Phe Thr Lys Arg Val Leu Gly Asp Ser Gly Glu Leu Asp Ile 1860 1865 1870 Leu Arg Gln Gln Met Glu Glu Arg Phe Val Ala Ser Asn Pro Ser Lys 1875 1880 1885 Val Ser Tyr Glu Pro Ile Thr Thr Thr Leu Arg Arg Lys Gln Glu Glu 1890 1895 1900 Val Ser Ala Val Val Leu Gln Arg Ala Tyr Arg Gly His Leu Ala Arg 1905 1910 1915 1920 Arg Gly Phe Ile Cys Arg Lys Met Ala Ser Asn Lys Leu Glu Asn Gly 1925 1930 1935 Gly Thr His Arg Asp Lys Lys Glu Ser Thr Pro Ser Thr Ala Ser Leu 1940 1945 1950 Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Lys Glu Lys Gln Gln Arg 1955 1960 1965 Ala Glu Glu Gly Arg Arg Glu Arg Ala Lys Arg Gln Lys Glu Val Arg 1970 1975 1980 Glu Ser Lys Cys 1985 696 base pairs nucleic acid single linear DNA (genomic) 5 CTCAACATGG TTACTATGAT GGTGGAGACA GACACTCAGA GCAAGCAGAT GGAGAACATT 60 CTTTACTGGA TTAATCTGGT CTTTGTCATC TTCTTCACCT GCGAGTGTGT GCTCAAAATG 120 TTTGCCTTGA GACACTACTA TTTCACCATT GGCTGGAACA TCTTTGACTT TGTGGTGGTC 180 ATCCTCTCCA TTGTGGGAAT GTTCCTGGCT GATATCATTG AGAAGTACTT CGTCTCCCCA 240 ACCCTATTCC GAGTTATCCG ATTGGCCCGT ATTGGGCGCA TCTTGCGTCT GATCAAGGGG 300 GCCAAAGGGA TCCGCACCCT GCTCTTTGGC CTTAATGATG TCGCTGGCCG CCCTGTTCAA 360 CATCGCCTCC TGCTCTTCCT CGTCATGTTC ATCTTCTCCA TTTTTGGCAT GTCCAACTTC 420 GCATACGTGA AGCACGAGGC CGGCATTGAC GACATGTTCA ACTTCGAGAC ATTTGGCAAC 480 AGCATGATCT GTTTGTTCCA GATCACAACG TCTGCTGGCT GGGATGGCCT GCTGCTGCCA 540 ATCCTGAACC GCCCCCCTGA CTGCAGCTTG GACAAAGAGC ACCCAGGGAG TGGCTTCAAA 600 GGGGACTGTG GGAACCCCTC GGTGGGCATC TTCTTCTTTG TGAGCTACAT CATCATCTCC 660 TTCCTGATTG TGGTGAACAT GTACATCGCA GTCATC 696 232 amino acids amino acid single linear peptide 6 Leu Asn Met Val Thr Met Met Val Glu Thr Asp Thr Gln Ser Lys Gln 1 5 10 15 Met Glu Asn Ile Leu Tyr Trp Ile Asn Leu Val Phe Val Ile Phe Phe 20 25 30 Thr Cys Glu Cys Val Leu Lys Met Phe Ala Leu Arg His Tyr Tyr Phe 35 40 45 Thr Ile Gly Trp Asn Ile Phe Asp Phe Val Val Val Ile Leu Ser Ile 50 55 60 Val Gly Met Phe Leu Ala Asp Ile Ile Glu Lys Tyr Phe Val Ser Pro 65 70 75 80 Thr Leu Phe Arg Val Ile Arg Leu Ala Arg Ile Gly Arg Ile Leu Arg 85 90 95 Leu Ile Lys Gly Ala Lys Gly Ile Arg Thr Leu Leu Phe Gly Leu Asn 100 105 110 Asp Val Ala Gly Arg Pro Val Gln His Arg Leu Leu Leu Phe Leu Val 115 120 125 Met Phe Ile Phe Ser Ile Phe Gly Met Ser Asn Phe Ala Tyr Val Lys 130 135 140 His Glu Ala Gly Ile Asp Asp Met Phe Asn Phe Glu Thr Phe Gly Asn 145 150 155 160 Ser Met Ile Cys Leu Phe Gln Ile Thr Thr Ser Ala Gly Trp Asp Gly 165 170 175 Leu Leu Leu Pro Ile Leu Asn Arg Pro Pro Asp Cys Ser Leu Asp Lys 180 185 190 Glu His Pro Gly Ser Gly Phe Lys Gly Asp Cys Gly Asn Pro Ser Val 195 200 205 Gly Ile Phe Phe Phe Val Ser Tyr Ile Ile Ile Ser Phe Leu Ile Val 210 215 220 Val Asn Met Tyr Ile Ala Val Ile 225 230 6556 base pairs nucleic acid single linear DNA (genomic) 7 CCAAGATGGC GCCCACCGCA GTCCCGCCCG CCGCAGCCTC GGCGCCTCTG CAGTCCGGCC 60 GCGCCTCCCG GGCCCCGCGC TAGGGCCGCT GCCGCCTCGC CCGCCGCCGC CGCCGCCAGC 120 TGACCTGTCC CGGACACATA ACTAACGAAG CTGCTGCAGG ATGAGAAGAT GGCAGCGCGG 180 CTGCTCGCAC CACCAGGCCC TGATAGTTTC AAGCCTTTCA CCCCTGAGTC GCTGGCAAAC 240 ATCGAGAGGC GTATTGCCGA GAGCAAGCTC AAGAAACCAC CAAAGGCGGA TGGCAGCCAC 300 CGGGAGGACG ATGAAGACAG CAAGCCCAAG CCAAACAGTG ACCTGGAGGC TGGGAAGAGT 360 TTGCCTTTCA TCTACGGGGA CATCCCGCAA GGCCTGGTTG CGGTTCCCCT GGAGGACTTT 420 GACCCTTACT ATTTGACGCA GAAAACCTTT GTAGTATTAA ACAGAGGGAA AACTCTCTTC 480 AGATTTAGTG CCACACCTGC CTTGTACATT TTAAGCCCTT TTAACCTGAT AAGAAGAATA 540 GCTATTAAAA TTTTGATACA CTCAGTTTTC AGCATGATCA TCATGTGCAC CATCCTGACC 600 AACTGTGTGT TCATGACCTT TAGTAACCCT CCAGAATGGT CCAAGAATGT GGAGTACACA 660 TTCACAGGGA TTTACACATT TGAATCACTA GTGAAAATCA TCGCAAGAGG TTTCTGCATA 720 GACGGCTTCA CCTTCTTGCG AGACCCGTGG AACTGGTTAG ACTTCAGTGT CATCATGATG 780 GCATATGTGA CAGAGTTTGT GGACCTGGGC AATGTCTCAG CGCTGAGAAC ATTCAGGGTT 840 CTCCGAGCTT TGAAAACTAT CTCTGTAATT CCAGGCCTGA AGACAATCGT GGGCGCCCTA 900 ATCCAGTCCG TGAAGAAGCT GTCGGACGTG ATGATCCTGA CAGTGTTCTG CCTGAGTGTT 960 TTCGCCCTGA TTGGCCTGCA GCTCTTCATG GGGAACCTTC GAAACAAGTG TGTCGTGTGG 1020 CCCATAAACT TCAACGAGAG CTACCTGGAG AACGGCACCA GAGGCTTTGA CTGGGAGGAA 1080 TATATCAACA ATAAAACAAA CTTTTACATG GTTCCTGGCA TGCTAGAACC CTTGCTCTGC 1140 GGGAACAGTT CTGATGCTGG GCAATGCCCA GAGGGATTCC AGTGCATGAA AGCAGGAAGG 1200 AACCCCAACT ACGGTTACAC CAGCTTTGAC ACCTTCAGCT GGGCCTTCTT GGCATTATTC 1260 CGCCTTATGA CCCAGGACTA TTGGGAGAAC TTATACCAGC TGACCTTACG AGCCGCTGGG 1320 AAAACGTACA TGATCTTCTT TGTCTTGGTC ATCTTCGTGG GTTCTTTCTA TCTGGTGAAC 1380 TTGATCTTGG CTGTGGTGGC CATGGCTTAT GAGGAACAGA ACCAGGCAAC ACTGGAGGAG 1440 GCAGAGCAAA AAGAGGCCGA GTTCAAGGCA ATGCTGGAGC AACTCAAGAA GCAGCAGGAG 1500 GAGGCACAGG CTGCTGCAAT GGCCACCTCA GCGGGCACTG TCTCGGAAGA CGCCATTGAA 1560 GAAGAAGGGG AAGATGGGGT AGGCTCTCCG AGGAGCTCTT CTGAACTGTC TAAACTCAGT 1620 TCCAAGAGCG CGAAGGAGCG GCGGAACCGA CGGAAGAAGA GGAAGCAGAA GGAGCTCTCT 1680 GAAGGCGAGG AGAAAGGGGA CCCGGAGAAG GTGTTTAAGT CAGAGTCGGA AGACGGTATG 1740 AGAAGGAAGG CCTTCCGGCT GCCAGACAAC AGGATAGGGA GGAAGTTTTC CATCATGAAT 1800 CAGTCGCTGC TCAGCATTCC AGGCTCGCCC TTCCTCTCCC GACATAACAG CAAAAGCAGC 1860 ATCTTCAGCT TCCGGGGACC CGGTCGGTTC CGGGACCCCG GCTCTGAGAA TGAGTTCGCA 1920 GACGATGAAC ACAGCACCGT GGAGGAGAGC GAGGGCCGGC GTGACTCGCT CTTCATCCCG 1980 ATCCGCGCCC GCGAGCGCCG CAGCAGCTAC AGTGGCTACA GCGGCTACAG CCAGTGCAGC 2040 CGCTCGTCGC GCATCTTCCC CAGCCTGCGG CGCAGCGTGA AGCGCAACAG CACGGTGGAC 2100 TGCAACGGCG TAGTGTCACT CATCGGGCCC GGCTCACACA TCGGGCGGCT CCTGCCTGAG 2160 GCAACGACTG AGGTGGAAAT TAAGAAGAAA GGCCCTGGAT CTCTTTTAGT TTCTATGGAC 2220 CAACTCGCCT CCTACGGACG GAAGGACAGA ATCAACAGCA TAATGAGCGT GGTCACAAAC 2280 ACGCTAGTGG AAGAGCTGGA AGAGTCTCAG AGAAAGTGCC CACCGTGCTG GTATAAGTTT 2340 GCCAACACTT TCCTCATCTG GGAGTGTCAC CCCTACTGGA TAAAACTGAA GGAGATCGTG 2400 AACTTAATCG TCATGGACCC TTTTGTAGAC TTAGCCATCA CCATCTGCAT CGTTCTGAAT 2460 ACGCTATTTA TGGCAATGGA GCACCATCCC ATGACACCAC AGTTCGAACA CGTCTTGGCC 2520 GTAGGAAATC TGGTGTTCAC CGGGATCTTC ACGGCGGAAA TGTTTCTGAA GCTCATAGCC 2580 ATGGACCCCT ACTATTATTT CCAAGAAGGC TGGAACATTT TTGACGGATT TATTGTCTCC 2640 CTCAGTTTAA TGGAGCTGAG TCTCGCAGAT GTGGAGGGGC TCTCAGTGCT GCGGTCTTTC 2700 CGACTGCTCC GAGTCTTCAA GCTGGCCAAG TCCTGGCCCA CCCTGAACAT GCTGATCAAG 2760 ATCATCGGGA ACTCCGTGGG TGCCCTGGGC AACCTGACCC TGGTGCTGGC CATCATCGTC 2820 TTCATCTTCG CCGTGGTGGG GATGCAGCTG TTTGGAAAGA GTTACAAGGA GTGCGTCTGT 2880 AAGATCAACC AGGAGTGCAA GCTCCCGCGC TGGCACATGA ACGACTTCTT CCACTCCTTC 2940 CTCATCGTCT TCCGAGTGCT GTGTGGGGAG TGGATCGAGA CCATGTGGGA CTGCATGGAG 3000 GTGGCCGGCC AGGCCATGTG CCTCATTGTC TTCATGATGG TTATGGTCAT TGGCAACCTG 3060 GTGGTGCTGA ATCTATTCCT GGCCTTGCTT CTGAGCTCCT TCAGCGCAGA CAACCTGGCG 3120 GCCACAGACG ACGACGGGGA AATGAACAAC CTGCAGATCT CAGTGATCCG GATCAAGAAG 3180 GGCGTGGCCT GGACCAAAGT GAAGGTGCAC GCCTTCATGC AGGCTCACTT CAAGCAGCGG 3240 GAGGCGGATG AAGTGAAACC CCTCGACGAG CTGTATGAGA AGAAGGCCAA CTGCATCGCC 3300 AACCACACGG GCGTGGATAT CCACCGGAAC GGCGACTTCC AGAAGAACGG GAACGGAACC 3360 ACCAGCGGCA TCGGCAGCAG CGTGGAGAAG TACATCATCG ACGAGGACCA CATGTCCTTC 3420 ATTAACAACC CAAACCTGAC CGTCCGGGTG CCCATTGCTG TGGGCGAGTC TGACTTCGAG 3480 AACCTCAACA CAGAGGATGT TAGCAGCGAA TCAGACCCTG AAGGCAGCAA AGATAAACTG 3540 GACGATACCA GCTCCTCAGA AGGAAGTACC ATCGACATCA AGCCTGAGGT GGAAGAAGTT 3600 CCCGTGGAGC AACCTGAGGA ATACTTGGAT CCGGACGCCT GCTTTACAGA GGGTTGCGTC 3660 CAGCGGTTCA AGTGCTGCCA GGTCAACATC GAGGAAGGAC TAGGCAAGTC GTGGTGGATC 3720 TTGCGGAAAA CCTGCTTCCT CATTGTGGAG CACAATTGGT TTGAGACCTT CATCATCTTC 3780 ATGATTCTGC TCAGCAGTGG CGCCCTGGCC TTTGAGGACA TCTACATTGA GCAGAGGAAG 3840 ACCATCCGCA CCATCCTGGA GTATGCGGAC AAGGTCTTCA CCTACATCTT CATCCTGGAG 3900 ATGTTGCTCA AGTGGACAGC CTACGGCTTC GTCAAGTTCT TCACCAATGC CTGGTGCTGG 3960 TTGGACTTCC TCATTGTGGC TGTCTCTTTA GTCAGCCTTA TAGCTAATGC CCTGGGCTAC 4020 TCGGAACTAG GTGCCATAAA GTCCCTTAGG ACCCTAAGAG CTTTGAGACC CTTAAGAGCC 4080 TTATCACGAT TTGAAGGGAT GAGGGTGGTG GTGAATGCCT TGGTGGGCGC CATCCCCTCC 4140 ATCATGAATG TGCTGCTGGT GTGTCTCATC TTCTGGCTGA TTTTCAGCAT CATGGGAGTT 4200 AACCTGTTTG CGGGGAAATA CCACTACTGC TTTAATGAGA CTTCTGAAAT CCGGTTCGAA 4260 ATCGATATTG TCAACAATAA AACGGACTGT GAGAAGCTCA TGGAGGGCAA CAGCACGGAG 4320 ATCCGATGGA AGAATGTCAA GATCAACTTT GACAATGTCG GAGCAGGGTA CCTGGCCCTT 4380 CTTCAAGTGG CAACCTTCAA AGGCTGGATG GACATCATGT ATGCGGCTGT AGATTCCCGA 4440 AAGCCAGACG AGCAGCCTGA CTACGAGGGC AACATCTACA TGTACATCTA CTTCGTCATC 4500 TTCATCATCT TCGGCTCCTT CTTCACCCTC AACCTGTTCA TCGGTGTCAT CATCGACAAC 4560 TTCAACCAGC AGAAGAAAAA GTTTGGAGGT CAGGACATCT TCATGACAGA GGAACAGAAG 4620 AAGTACTACA ATGCCATGAA AAAGCTGGGC TCCAAGAAGC CACAGAAGCC CATCCCCCGA 4680 CCCTTGAACA AAATCCAAGG GATTGTCTTT GATTTCGTCA CTCAACAAGC CTTTGACATT 4740 GTGATCATGA TGCTCATCTG CCTTAACATG GTGACAATGA TGGTGGAGAC AGACACTCAG 4800 AGCAAGCAGA TGGAGAACAT TCTTTACTGG ATTAATCTGG TCTTTGTCAT CTTCTTCACC 4860 TGCGAGTGTG TGCTCAAAAT GTTTGCCTTG AGACACTACT ATTTCACCAT TGGCTGGAAC 4920 ATCTTTGACT TTGTGGTGGT CATCCTCTCC ATTGTGGGAA TGTTCCTGGC TGATATCATT 4980 GAGAAGTACT TCGTCTCCCC AACCCTATTC CGAGTTATCC GATTGGCCCG TATTGGGCGC 5040 ATCTTGCGTC TGATCAAGGG CGCCAAAGGG ATCCGCACCC TGCTCTTTGC CTTAATGATG 5100 TCGCTGCCCG CCCTGTTCAA CATCGGCCTC CTGCTCTTCC TCGTCATGTT CATCTTCTCC 5160 ATTTTTGGCA TGTCCAACTT CGCATACGTG AAGCACGAGG CCGGCATTGA CGACATGTTC 5220 AACTTCGAGA CATTTGGCAA CAGCATGATC TGTTTGTTCC AGATCACAAC GTCTGCTGGC 5280 TGGGATGGCC TGCTGCTGCC AATCCTGAAC CGCCCCCCTG ACTGCAGCTT GGACAAAGAG 5340 CACCCAGGGA GTGGCTTCAA AGGGGACTGT GGGAACCCCT CGGTGGGCAT CTTCTTCTTT 5400 GTGAGCTACA TCATCATCTC CTTCCTGATT GTGGTGAACA TGTACATCGC CATCATCCTG 5460 GAGAACTTCA GCGTGGCCAC CGAGGAGAGC GCCGACCCTC TGAGTGAGGA TGACTTCGAG 5520 ACTTTCTATG AGATCTGGGA GAAGTTTGAC CCAGACGCCA CCCAGTTCAT CGAGTACTGT 5580 AAGCTGGCAG ACTTTGCCGA CGCCCTGGAG CACCCGCTCC GAGTACCCAA GCCCAACACC 5640 ATCGAGCTCA TCGCCATGGA CCTGCCCATG GTGAGCGGAG ATCGCATCCA CTGCTTGGAC 5700 ATCCTTTTCG CCTTCACCAA GCGAGTCCTG GGAGACAGTG GGGAGTTGGA CATCCTGCGG 5760 CAGCAGATGG AGGAGCGGTT CGTGGCATCC AATCCTTCCA AAGTGTCTTA CGAGCCTATC 5820 ACAACCACTC TGCGGCGCAA GCAGGAGGAG GTGTCTGCAG TGGTCCTGCA GCGTGCCTAC 5880 AGGGGACACT TGGCTAGGCG GGGCTTCATC TGCAGAAAGA TGGCCTCCAA CAAGCTGGAG 5940 AATGGAGGCA CACACAGAGA CAAGAAGGAG AGCACCCCGT CCACAGCCTC CCTCCCCTCT 6000 TACGACAGCG TCACAAAGCC AGACAAGGAG AAGCAGCAGC GTGCGGAGGA GGGCAGAAGG 6060 GAAAGAGCCA AGAGGCAAAA AGAGGTCAGG GAGTCCAAGT GCTAGAGGAG GGGAAAGGAA 6120 GCTTACCCCG GCTGAACACT GGCAAGTGAA AGCTTGTTTA CAAACTTCCG AATCTCACGG 6180 ATGCAGAGCA GCTGTGCAGA CGCTCGCTGT ACTGGAAGAC CTATACCAAA CATAGTCTGC 6240 TTACATGTGA CATGGTGGCA TCCTGAGCGG TGACTGCTGG GGACAAAGGA CCCTGCTCCC 6300 TGGACTCACA GATCTCCTAT CGCTTGGGCA GACGGTTACT GCATGTTCCA CACTTAGTCA 6360 ATGCAACTTA GGACTAAACT AACCAGGATA CAAAACCGAG GCGGCTGCCG GGACCAGCAG 6420 ATCACCGCTG CAGCCAAATG GATTTTATTT TTTCATTTTG TTGATTCTCA GAAGCAGAAA 6480 GCATCACTTT AAAAGTTTGT TTGTTCATNC AAACAATATT TGAATTCTTA CATTAGTTAA 6540 GCTAAGCANC AAAAAG 6556 6826 base pairs nucleic acid single linear DNA (genomic) 8 ATGAGAAGAT CGGCGCGGCT GCTCGCACCA CCAGGCCCTG ATAGTTTCAA GCCTTTCACC 60 CCTGAGTCGC TGGCAAACAT CGAGAGGCGT ATTGCCGAGA GCAAGCTCAA GAAACCACCA 120 AAGGCGGATG GCAGCCACCG GGAGGACGAT GAAGACAGCA AGCCCAAGCC AAACAGTGAC 180 CTGGAGGCTG GGAAGAGTTT GCCTTTCATC TACGGGGACA TCCCGCAAGG CCTGGTTGCG 240 GTTCCCCTGG AGGACTTTGA CCCTTACTAT TTGACGCAGA AAACCTTTGT AGTATTAAAC 300 AGAGGGAAAA CTCTCTTCAG ATTTAGTGCC ACACCTGCCT TGTACATTTT AAGCCCTTTT 360 AACCTGATAA GAAGAATAGC TATTAAAATT TTGATACACT CAGTTTTCAG CATGATCATC 420 ATGTGCACCA TCCTGACCAA CTGTGTGTTC ATGACCTTTA GTAACCCTCC AGAATGGTCC 480 AAGAATGTGG AGTACACATT CACAGGGATT TACACATTTG AATCACTAGT GAAAATCATC 540 GCAAGAGGTT TCTGCATAGA CGGCTTCACC TTCTTACGAG ACCCGTGGAA CTGGTTAGAC 600 TTCAGTGTCA TCATGATGGC ATATGTGACA GAGTTTGTGG ACCTGGGCAA TGTCTCAGCG 660 CTGAGAACAT TCAGGGTTCT CCGAGCTTTG AAAACTATCT CTGTAATTCC AGGCCTGAAG 720 ACAATCGTGG GCGCCCTAAT CCAGTCCGTG AAGAAGCTGT CGGACGTGAT GATCCTGACA 780 GTGTTCTGCC TGAGTGTTTT CGCCCTGATT GGCCTGCAGC TCTTTCATGG GAACCTTTCG 840 AAACAGTGTG TCGTGTGGCC CATAAACTTC AACGAGAGCT ACCTGGAGAA CGGCACCAGA 900 GGCTTTGACT GGGAGGAATA TATCAACAAT AAAACAAACT TTTACATGGT TCCTGGCATG 960 CTAGAACCCT TGCTCTGCGG GAACAGTTCT GATGCTGGGC AATGCGAAGG ATTCCAGTGC 1020 AGTAAAGCAG GAAGGAACCC CAACTACGGT TACACCAGCT TTGACACCTT CAGCTGGGCC 1080 TTCTTGGCAT TATTCCGCCT TATGACCCAG GACTATTGGG AGAACTTATA CCAGCTGACC 1140 TTACGAGCCG CTGGGAAAAC GTACATGATC TTCTTTGTCT TGGTCATCTT CGTGGGTTCT 1200 TTCTATCCGG TGAACTTGAT CTTGGCTGTG GTGGCCATGG CTTATGAGGA ACAGAACCAG 1260 GCAACACTGG AGGAGGCAGA GCAAAAAGAG GCCGAGTTCA AGGCAATGCT GGAGCAACTC 1320 AAGAAGCAGC AGGAGGAGGC ACAGGCTGCT GCAATGGCCA CCTCAGCGGG CACTGTCTCG 1380 GAAGACGCCA TTGAAGAAGA AGGGGAAGAT GGGGTAGGCT CTCCGAGGAG CTCTTCTGAA 1440 CTGTCTAAAC TCAGTTCCAA GAGCGCGAAG GAGCGGCGGA ACCGACGGAA GAAGAGGAAG 1500 CAGAAGGAGC TCTCTGAAGG CGAGGAGAAA GGGGACCCGG AGAAGGTGTT TAAGTCAGAG 1560 TCGGAATACG GTATGAGAAG GAAGGCCTTC CGGCTGCCAG ACAACAGGAT AGGGAGGAAG 1620 TTTTCCATCA TGAATCAGTC GCTGCTCAGC ATTCCAGGCT CGCCCTTCCT CTCCCGACAT 1680 AACAGCAAAA GCAGCATCTT CAGCTTCGGG GACCCGTCGG TTCGGGACCC CGGCTCTGAG 1740 AATGAGTTCG CAGACGATGA ACACAGCACC GTGGAGGAGA GCGAGGGCCG GCGTGACTCG 1800 CTCTTCATCC CGATCCGCGC CCGCGAGCGC CGCAGCAGCT ACAGTGGCTA CAGCGGCTAC 1860 AGCCAGTGCA GCCGCTCGTC GCGCATCTCC CCAGCCTGCG CGCAGCGTGA AGCCAACAGC 1920 ACGGTGGACT GCAACGGCGT AGTGTCACTC ATCGGGCCCG GCTCACACAT CGGGCGGCTC 1980 CTGCTGAGGC AACGACTGAG GTGGAAATTA AGAAGAAAGG CCCTGGACTC TTTTAGTTTC 2040 TATGGACCAA CTCGCCTCCT ACGGACGGAA GGACAGAATC AACAGCATAA TGAGCGTGGT 2100 CACAAACACG CTAGTGAAGA GCTGGAAGAG TCTCAGAGAA AGTGCCCACC GTGCTGGTAT 2160 AAGTTTGCCA ACACTTTCCT CATCTGGGAG TGTCACCCCT ACTGGATAAA ACTGAAGGAG 2220 ATCGTGAACT TAATCGTCAT GGACCCTTTT GTAGACTTAG CCATCACCAT CTGCATCGTT 2280 CTGAATACGC TATTTATGGC AATGGAGCAC CATCCCATGA CACCACAGTT CGAACACGTC 2340 TTGGCCGTAG GAAATCTGGT GTTCACCGGG ATCTTCACGG CGGAAATGTT TCTGAAGCTC 2400 ATAGCCATGG ACCCCTACTA TTATTTCCAA GAAGGCTGGA ACATTTTTGA CGGATTTATT 2460 GTCTCCCTCA GTTTAATGGA GCTGAGTCTC GCAGATGTGG AGGGGCTCTC AGTGCTGCGG 2520 TCTTTCCGAC TGCTCCGAGT CTTCAAGCTG GCCAAGTCCT GGCCCACCCT GAACATGCTG 2580 ATCAAGATCA TCGGGAACTC CGTGGGTGCC CTGGGCAACC TGACCCTGGT GCTGGCCATC 2640 ATCGTCTTCA TCTTCGCCGT GGTGGGGATG CAGCTGTTTG GAAAGAGTTA CAAGGAGTGC 2700 GTCTGTAAGA TCAACCAGGA GTGCAAGCTC CCGCGCTGGC ACATGAACGA CTTCTTCCAC 2760 TCCTTCCTCA TCGTCTTCCG AGTGCTGTGT GGGGAGTGGA TCGAGACCAT GTGGGACTGC 2820 ATGGAGGTGG CCGGCCAGGC CATGTGCCTC ATTGTCTTCA TGATGGTTAT GGTCATTGGC 2880 AACCTGGTGG TGCTGAATCT ATTCCTGGCC TTGCTTCTGA GCTCCTTCAG CGCAGACAAC 2940 CTGGCGGCCA CAGACGACGA CGGGGAAATG AACAACCTGC AGATCTCAGT GATCCGGATC 3000 AAGAAGGGCG TGGCCTGGAC CAAAGTGAAG GTGCACGCCT TCATGCAGGC TCACTTCAAG 3060 CAGCGGGAGG CGGATGAAGT GAAACCCCTC GACGAGCTGT ATGAGAAGAA GGCCAACTGC 3120 ATCGCCAACC ACACGGGCGT GGATATCCAC CGGAACGGCG ACTTCCAGAA GAACGGGAAC 3180 GGAACCACCA GCGGCATCGG CAGCAGCGTG GAGAAGTACA TCATCGACGA GGACCACATG 3240 TCCTTCATTA ACAACCCAAA CCTGACCGTC CGGGTGCCCA TTGCTGTGGG CGAGTCTGAC 3300 TTCGAGAACC TCAACACAGA GGATGTTAGC AGCGAATCAG ACCCTGAAGG CAGCAAAGAT 3360 AAACTGGACG ATACCAGCTC CTCAGAAGGA AGTACCATCG ACATCAAGCC TGAGGTGGAA 3420 GAAGTTCCCG TGGAGCAACC TGAGGAATAC TTGGATCCGG ACGCCTGCTT TACAGAGGGT 3480 TGCGTCCAGC GGTTCAAGTG CTGCCAGGTC AACATCGAGG AAGGACTAGG CAAGTCGTGG 3540 TGGATCTTGC GGAAAACCTG CTTCCTCATT GTGGAGCACA ATTGGTTTGA GACCTTCATC 3600 ATCTTCATGA TTCTGCTCAG CAGTGGCGCC CTGGCCTTTG AGGACATCTA CATTGAGCAG 3660 AGGAAGACCA TCCGCACCAT CCTGGAGTAT GCGGACAAGG TCTTCACCTA CATCTTCATC 3720 CTGGAGATGT TGCTCAAGTG GACCACGTAC GGCTTCGTCA AGTTCTTCAC CAATGCCTGG 3780 TGCTGGTTGG ACTTCCTCAT TGTGGCTGTC TCTTTAGTCA GCCTTATAGC TAATGCCCTG 3840 GGCTACTCGG AACTAGGTGC CATAAAGTCC CTTAGGACCC TAAGAGCTTT GAGACCCTTA 3900 AGAGCCTTAT CACGATTTGA AGGGATGAGG GTGGTGGTGA ATGCCTTGGT GGGTGCCATC 3960 CCCTCCATCA TGAATGTGCT GCTGGTGTGT CTCATCTTCT GGCTGATTTT CAGCATCATG 4020 GGAGTTAACC TGTTTGCGGG GAAATACCAC TACTGCTTTA ATGAGACTTC TGAAATCCGG 4080 TTCGAAATCG ATATTGTCAA CAATAAAACG GACTGTGAGA AGCTCATGGA GGGCAACAGC 4140 ACGGAGATCC GATGGAAGAA TGTCAAGATC AACTTTGACA ATGTCGGAGC AGGGTACCTG 4200 GCCCTTCTTC AAGTGGCAAC CTTCAAAGGC TGGATGGACA TCATGTATGC GGCTGTAGAT 4260 TCCCGAAAGC CAGACGAGCA GCCTGACTAC GAGGGCAACA TCTACATGTA CATCTACTTC 4320 GTCATCTTCA TCATCTTCGG CTCCTTCTTC ACCCTCAACC TGTTCATCGG TGTCATCATC 4380 GACAACTTCA ACCAGCAGAA GAAAAAGTTT GGAGGTCAGG ACATCTTCAT GACAGAGGAA 4440 CAGAAGAAGT ACTATAATGC CATGAAAAAG CTGGGCTCCA AGAAGCCACA GAAGCCCATC 4500 CCCCGACCCT TGAACAAAAT CCAAGGGATT GTCTTTGATT TCGTCACTCA ACAAGCCTTT 4560 GACATTGTGA TCATGATGCT CATCTGCCTT AACATGGTGA CAATGATGGT GGAGACAGAC 4620 ACTCAGAGCA AGCAGATGGA GAACATTCTT TACTGGATTA ATCTGGTCTT TGTCATCTTC 4680 TTCACCTGCG AGTGTGTGCT CAAAATGTTT GCCTTGAGAC ACTACTACTT CACCATTGGC 4740 TGGAACATCT TTGACTTTGT GGTGGTCATC CTCTCCATTG TGGGAATGTT CCTGGCTGAT 4800 ATCATTGAGA AGTACTTCGT CTCCCCAACC CTATTCCGAG TTATCCGATT GGCCCGTATT 4860 GGGCGCATCT TGCGTCTGAT CAAGGGCGCC AAAGGGATCC GCACTCTGCT CTTTGCTCTG 4920 ATGATGTCGC TGCCCGCCCT GTTCAACATC GGCCTCCTGC TCTTCCTCGT CATGTTCATC 4980 TTCTCCATTT TTGGCATGTC CAACTTCGCA TACGTGAAGC ACGAGGCCGG CATTGACGAC 5040 ATGTTCAACT TCGAGACATT TGGCAACAGC ATGATCTGTT TGTTCCAGAT CACAACGTCT 5100 GCTGGCTGGG ATGGCCTGCT GCTGCCAATC CTGAACCGCC CCCCTGACTG CAGCTTGGAC 5160 AAAGAGCACC CAGGGAGTGG CTTCAAAGGG GACTGTGGGA ACCCCTCGGT GGGCATCTTC 5220 TTCTTTGTGA GCTACATCAT CATCTCCTTC CTGATTGTGG TGAACATGTG CATCGCCATC 5280 ATCCTGGAGA ACTTCAGCGT GGCCACCGAG GAGAGCGCCG ACCCTCTGAG TGAGGATGAC 5340 TTCGAGACTT TCTATGAGAT CTGGGAGAAG TTTGACCCAG ACGCCACCCA GTTCATCGAG 5400 TACTGTAAGC TGGCAGACTT TGCCGACGCC CTGGAGCACC CGCTCCGAGT ACCCAAGCCC 5460 AACACCATCG AGCTCATCGC CATGGACCTG CCCATGGTGA GCGGAGATCG CATCCACTGC 5520 TTGGACATCC TTTTCGCCTT CACCAAGGCA GTCCTGGGAG ACAGTGGGGA GTTGGACATC 5580 CTGCGGCAGC AGATGGAGGA GCGGTTCGTG GCATCCAATC CTTCCAAAGT GTCTTACGAA 5640 GCCTATCACA CCACTCTGCG GCGCAACGAG GAGGAGGTGT CTGCAGTGGT CCTGCAGCGT 5700 GCCTACAGGG GACACTTGGC TAGGCGGGGC TTCATCTGCA GAAAGATGGC CTCCAACAAG 5760 CTGGAGAATG GAGGCACACA CAGAGACAAG AAGGAGAGCA CCCCGTCCAC AGCCTCCCTC 5820 CCCTCTTACG ACAGCGTCAC AAAGCCAGAC AAGGAGAAGC AGCAGCGTGC GGAGGAGGGC 5880 AGAAGGGAAA GAGCCAAGAG GCAAAAAGAG GTCAGGGAGT CCAAGTGCTA GAGGAGGGGA 5940 AAGGAAGCTT ACCCCGGCTG AACACTGGCA AGTGAAAGCT TGTTTACAAA CTTCCGAATC 6000 TCACGGATGC AGACAGCTGT GCAGACGCTC GCTGTACTGG AAGACCTATA CCAAACATAG 6060 TCTGCTTACA TGTGACATGG TGGCATCCTG AGCGGTGACT GCTGCTGGGG ACAAAGGACC 6120 CTGCTCCCTG GACTCACAGA TCTCCTATCG CTTGGGCAGA CGGTTACTGC ATGTTCCACA 6180 CTTAGTCAAT GCAACTTAGG ACTAAACTAA CCAGGATACA AAACCGAGGC GGCTGGCGAC 6240 CAGCAGATCA CCGCTGCAGC CAAATGGATT TTATTTTTTC ATTTTGTTGA TTCTCAGAAG 6300 CAGAAAGCAT CACTTTAAAA GTTTGTTTGT TCATGCAAAC AATATTTGAA TTCTTACATT 6360 AGTTAAGCTA AGCAGCAAAA AGAAACACAC ACGCACACAG ACACACAAAG ACACACACAC 6420 ATTCAGCCTA TGTCACTAAT CGTCTGTTTC TTTAACATAA CAGCATCTTC TCCACACGAG 6480 CGGCACGTGG TTTGGAGATG GGTGGGGGAA AATCAGGGTT TCAGGCTGAG GAGGACTTGC 6540 TCAGGCCAAT CCCAAATATG TGCTCGTTCA ATGCATAGAA GTGACCTGCA TGATGGCATG 6600 CTGTGTTCAG AAGTCATGCA TGAGACCCAC ACACCACAAG ACACTAGTAC TCCTGTNNCC 6660 ATCCACAGGC TCAGCCTGCG GACAGGACCA GCCCTGCACC GTTCACTGTA TTTGGAGAAA 6720 TGGTAAGAGT TCCACACCGG CTGCAGTCCT CTCAGTGTAG GATTCTTTCG TACACCTCTG 6780 GGTAGGGAGA CATAATTAAC CAATTGACCA CTACCAACAA AACAAT 6826 1976 amino acids amino acid single linear peptide 9 Met Arg Arg Ser Ala Arg Leu Leu Ala Pro Pro Gly Pro Asp Ser Phe 1 5 10 15 Lys Pro Phe Thr Pro Glu Ser Leu Ala Asn Ile Glu Arg Arg Ile Ala 20 25 30 Glu Ser Lys Leu Lys Lys Pro Pro Lys Ala Asp Gly Ser His Arg Glu 35 40 45 Asp Asp Glu Asp Ser Lys Pro Lys Pro Asn Ser Asp Leu Glu Ala Gly 50 55 60 Lys Ser Leu Pro Phe Ile Tyr Gly Asp Ile Pro Gln Gly Leu Val Ala 65 70 75 80 Val Pro Leu Glu Asp Phe Asp Pro Tyr Tyr Leu Thr Gln Lys Thr Phe 85 90 95 Val Val Leu Asn Arg Gly Lys Thr Leu Phe Arg Phe Ser Ala Thr Pro 100 105 110 Ala Leu Tyr Ile Leu Ser Pro Phe Asn Leu Ile Arg Arg Ile Ala Ile 115 120 125 Lys Ile Leu Ile His Ser Val Phe Ser Met Ile Ile Met Cys Thr Ile 130 135 140 Leu Thr Asn Cys Val Phe Met Thr Phe Ser Asn Pro Pro Glu Trp Ser 145 150 155 160 Lys Asn Val Glu Tyr Thr Phe Thr Gly Ile Tyr Thr Phe Glu Ser Leu 165 170 175 Val Lys Ile Ile Ala Arg Gly Phe Cys Ile Asp Gly Phe Thr Phe Leu 180 185 190 Arg Asp Pro Trp Asn Trp Leu Asp Phe Ser Val Ile Met Met Ala Tyr 195 200 205 Val Thr Glu Phe Val Asp Leu Gly Asn Val Ser Ala Leu Arg Thr Phe 210 215 220 Arg Val Leu Arg Ala Leu Lys Thr Ile Ser Val Ile Pro Gly Leu Lys 225 230 235 240 Thr Ile Val Gly Ala Leu Ile Gln Ser Val Lys Lys Leu Ser Asp Val 245 250 255 Met Ile Leu Thr Val Phe Cys Leu Ser Val Phe Ala Leu Ile Gly Leu 260 265 270 Gln Leu Phe His Gly Asn Leu Ser Lys Gln Cys Val Val Trp Pro Ile 275 280 285 Asn Phe Asn Glu Ser Tyr Leu Glu Asn Gly Thr Arg Gly Phe Asp Trp 290 295 300 Glu Glu Tyr Ile Asn Asn Lys Thr Asn Phe Tyr Met Val Pro Gly Met 305 310 315 320 Leu Glu Pro Leu Leu Cys Gly Asn Ser Ser Asp Ala Gly Gln Cys Glu 325 330 335 Gly Phe Gln Cys Ser Lys Ala Gly Arg Asn Pro Asn Tyr Gly Tyr Thr 340 345 350 Ser Phe Asp Thr Phe Ser Trp Ala Phe Leu Ala Leu Phe Arg Leu Met 355 360 365 Thr Gln Asp Tyr Trp Glu Asn Leu Tyr Gln Leu Thr Leu Arg Ala Ala 370 375 380 Gly Lys Thr Tyr Met Ile Phe Phe Val Leu Val Ile Phe Val Gly Ser 385 390 395 400 Phe Tyr Pro Val Asn Leu Ile Leu Ala Val Val Ala Met Ala Tyr Glu 405 410 415 Glu Gln Asn Gln Ala Thr Leu Glu Glu Ala Glu Gln Lys Glu Ala Glu 420 425 430 Phe Lys Ala Met Leu Glu Gln Leu Lys Lys Gln Gln Glu Glu Ala Gln 435 440 445 Ala Ala Ala Met Ala Thr Ser Ala Gly Thr Val Ser Glu Asp Ala Ile 450 455 460 Glu Glu Glu Gly Glu Asp Gly Val Gly Ser Pro Arg Ser Ser Ser Glu 465 470 475 480 Leu Ser Lys Leu Ser Ser Lys Ser Ala Lys Glu Arg Arg Asn Arg Arg 485 490 495 Lys Lys Arg Lys Gln Lys Glu Leu Ser Glu Gly Glu Glu Lys Gly Asp 500 505 510 Pro Glu Lys Val Phe Lys Ser Glu Ser Glu Tyr Gly Met Arg Arg Lys 515 520 525 Ala Phe Arg Leu Pro Asp Asn Arg Ile Gly Arg Lys Phe Ser Ile Met 530 535 540 Asn Gln Ser Leu Leu Ser Ile Pro Gly Ser Pro Phe Leu Ser Arg His 545 550 555 560 Asn Ser Lys Ser Ser Ile Phe Ser Phe Gly Asp Pro Ser Val Arg Asp 565 570 575 Pro Gly Ser Glu Asn Glu Phe Ala Asp Asp Glu His Ser Thr Val Glu 580 585 590 Glu Ser Glu Gly Arg Arg Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg 595 600 605 Glu Arg Arg Ser Ser Tyr Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser 610 615 620 Arg Ser Ser Arg Ile Ser Pro Ala Cys Ala Gln Arg Glu Ala Asn Ser 625 630 635 640 Thr Val Asp Cys Asn Gly Val Val Ser Leu Ile Gly Pro Gly Ser His 645 650 655 Ile Gly Arg Leu Leu Leu Arg Gln Arg Leu Arg Trp Lys Leu Arg Arg 660 665 670 Lys Ala Leu Asp Ser Phe Ser Phe Tyr Gly Pro Thr Arg Leu Leu Arg 675 680 685 Thr Glu Gly Gln Asn Gln Gln His Asn Glu Arg Gly His Lys His Ala 690 695 700 Ser Glu Glu Leu Glu Glu Ser Gln Arg Lys Cys Pro Pro Cys Trp Tyr 705 710 715 720 Lys Phe Ala Asn Thr Phe Leu Ile Trp Glu Cys His Pro Tyr Trp Ile 725 730 735 Lys Leu Lys Glu Ile Val Asn Leu Ile Val Met Asp Pro Phe Val Asp 740 745 750 Leu Ala Ile Thr Ile Cys Ile Val Leu Asn Thr Leu Phe Met Ala Met 755 760 765 Glu His His Pro Met Thr Pro Gln Phe Glu His Val Leu Ala Val Gly 770 775 780 Asn Leu Val Phe Thr Gly Ile Phe Thr Ala Glu Met Phe Leu Lys Leu 785 790 795 800 Ile Ala Met Asp Pro Tyr Tyr Tyr Phe Gln Glu Gly Trp Asn Ile Phe 805 810 815 Asp Gly Phe Ile Val Ser Leu Ser Leu Met Glu Leu Ser Leu Ala Asp 820 825 830 Val Glu Gly Leu Ser Val Leu Arg Ser Phe Arg Leu Leu Arg Val Phe 835 840 845 Lys Leu Ala Lys Ser Trp Pro Thr Leu Asn Met Leu Ile Lys Ile Ile 850 855 860 Gly Asn Ser Val Gly Ala Leu Gly Asn Leu Thr Leu Val Leu Ala Ile 865 870 875 880 Ile Val Phe Ile Phe Ala Val Val Gly Met Gln Leu Phe Gly Lys Ser 885 890 895 Tyr Lys Glu Cys Val Cys Lys Ile Asn Gln Glu Cys Lys Leu Pro Arg 900 905 910 Trp His Met Asn Asp Phe Phe His Ser Phe Leu Ile Val Phe Arg Val 915 920 925 Leu Cys Gly Glu Trp Ile Glu Thr Met Trp Asp Cys Met Glu Val Ala 930 935 940 Gly Gln Ala Met Cys Leu Ile Val Phe Met Met Val Met Val Ile Gly 945 950 955 960 Asn Leu Val Val Leu Asn Leu Phe Leu Ala Leu Leu Leu Ser Ser Phe 965 970 975 Ser Ala Asp Asn Leu Ala Ala Thr Asp Asp Asp Gly Glu Met Asn Asn 980 985 990 Leu Gln Ile Ser Val Ile Arg Ile Lys Lys Gly Val Ala Trp Thr Lys 995 1000 1005 Val Lys Val His Ala Phe Met Gln Ala His Phe Lys Gln Arg Glu Ala 1010 1015 1020 Asp Glu Val Lys Pro Leu Asp Glu Leu Tyr Glu Lys Lys Ala Asn Cys 1025 1030 1035 1040 Ile Ala Asn His Thr Gly Val Asp Ile His Arg Asn Gly Asp Phe Gln 1045 1050 1055 Lys Asn Gly Asn Gly Thr Thr Ser Gly Ile Gly Ser Ser Val Glu Lys 1060 1065 1070 Tyr Ile Ile Asp Glu Asp His Met Ser Phe Ile Asn Asn Pro Asn Leu 1075 1080 1085 Thr Val Arg Val Pro Ile Ala Val Gly Glu Ser Asp Phe Glu Asn Leu 1090 1095 1100 Asn Thr Glu Asp Val Ser Ser Glu Ser Asp Pro Glu Gly Ser Lys Asp 1105 1110 1115 1120 Lys Leu Asp Asp Thr Ser Ser Ser Glu Gly Ser Thr Ile Asp Ile Lys 1125 1130 1135 Pro Glu Val Glu Glu Val Pro Val Glu Gln Pro Glu Glu Tyr Leu Asp 1140 1145 1150 Pro Asp Ala Cys Phe Thr Glu Gly Cys Val Gln Arg Phe Lys Cys Cys 1155 1160 1165 Gln Val Asn Ile Glu Glu Gly Leu Gly Lys Ser Trp Trp Ile Leu Arg 1170 1175 1180 Lys Thr Cys Phe Leu Ile Val Glu His Asn Trp Phe Glu Thr Phe Ile 1185 1190 1195 1200 Ile Phe Met Ile Leu Leu Ser Ser Gly Ala Leu Ala Phe Glu Asp Ile 1205 1210 1215 Tyr Ile Glu Gln Arg Lys Thr Ile Arg Thr Ile Leu Glu Tyr Ala Asp 1220 1225 1230 Lys Val Phe Thr Tyr Ile Phe Ile Leu Glu Met Leu Leu Lys Trp Thr 1235 1240 1245 Thr Tyr Gly Phe Val Lys Phe Phe Thr Asn Ala Trp Cys Trp Leu Asp 1250 1255 1260 Phe Leu Ile Val Ala Val Ser Leu Val Ser Leu Ile Ala Asn Ala Leu 1265 1270 1275 1280 Gly Tyr Ser Glu Leu Gly Ala Ile Lys Ser Leu Arg Thr Leu Arg Ala 1285 1290 1295 Leu Arg Pro Leu Arg Ala Leu Ser Arg Phe Glu Gly Met Arg Val Val 1300 1305 1310 Val Asn Ala Leu Val Gly Ala Ile Pro Ser Ile Met Asn Val Leu Leu 1315 1320 1325 Val Cys Leu Ile Phe Trp Leu Ile Phe Ser Ile Met Gly Val Asn Leu 1330 1335 1340 Phe Ala Gly Lys Tyr His Tyr Cys Phe Asn Glu Thr Ser Glu Ile Arg 1345 1350 1355 1360 Phe Glu Ile Asp Ile Val Asn Asn Lys Thr Asp Cys Glu Lys Leu Met 1365 1370 1375 Glu Gly Asn Ser Thr Glu Ile Arg Trp Lys Asn Val Lys Ile Asn Phe 1380 1385 1390 Asp Asn Val Gly Ala Gly Tyr Leu Ala Leu Leu Gln Val Ala Thr Phe 1395 1400 1405 Lys Gly Trp Met Asp Ile Met Tyr Ala Ala Val Asp Ser Arg Lys Pro 1410 1415 1420 Asp Glu Gln Pro Asp Tyr Glu Gly Asn Ile Tyr Met Tyr Ile Tyr Phe 1425 1430 1435 1440 Val Ile Phe Ile Ile Phe Gly Ser Phe Phe Thr Leu Asn Leu Phe Ile 1445 1450 1455 Gly Val Ile Ile Asp Asn Phe Asn Gln Gln Lys Lys Lys Phe Gly Gly 1460 1465 1470 Gln Asp Ile Phe Met Thr Glu Glu Gln Lys Lys Tyr Tyr Asn Ala Met 1475 1480 1485 Lys Lys Leu Gly Ser Lys Lys Pro Gln Lys Pro Ile Pro Arg Pro Leu 1490 1495 1500 Asn Lys Ile Gln Gly Ile Val Phe Asp Phe Val Thr Gln Gln Ala Phe 1505 1510 1515 1520 Asp Ile Val Ile Met Met Leu Ile Cys Leu Asn Met Val Thr Met Met 1525 1530 1535 Val Glu Thr Asp Thr Gln Ser Lys Gln Met Glu Asn Ile Leu Tyr Trp 1540 1545 1550 Ile Asn Leu Val Phe Val Ile Phe Phe Thr Cys Glu Cys Val Leu Lys 1555 1560 1565 Met Phe Ala Leu Arg His Tyr Tyr Phe Thr Ile Gly Trp Asn Ile Phe 1570 1575 1580 Asp Phe Val Val Val Ile Leu Ser Ile Val Gly Met Phe Leu Ala Asp 1585 1590 1595 1600 Ile Ile Glu Lys Tyr Phe Val Ser Pro Thr Leu Phe Arg Val Ile Arg 1605 1610 1615 Leu Ala Arg Ile Gly Arg Ile Leu Arg Leu Ile Lys Gly Ala Lys Gly 1620 1625 1630 Ile Arg Thr Leu Leu Phe Ala Leu Met Met Ser Leu Pro Ala Leu Phe 1635 1640 1645 Asn Ile Gly Leu Leu Leu Phe Leu Val Met Phe Ile Phe Ser Ile Phe 1650 1655 1660 Gly Met Ser Asn Phe Ala Tyr Val Lys His Glu Ala Gly Ile Asp Asp 1665 1670 1675 1680 Met Phe Asn Phe Glu Thr Phe Gly Asn Ser Met Ile Cys Leu Phe Gln 1685 1690 1695 Ile Thr Thr Ser Ala Gly Trp Asp Gly Leu Leu Leu Pro Ile Leu Asn 1700 1705 1710 Arg Pro Pro Asp Cys Ser Leu Asp Lys Glu His Pro Gly Ser Gly Phe 1715 1720 1725 Lys Gly Asp Cys Gly Asn Pro Ser Val Gly Ile Phe Phe Phe Val Ser 1730 1735 1740 Tyr Ile Ile Ile Ser Phe Leu Ile Val Val Asn Met Cys Ile Ala Ile 1745 1750 1755 1760 Ile Leu Glu Asn Phe Ser Val Ala Thr Glu Glu Ser Ala Asp Pro Leu 1765 1770 1775 Ser Glu Asp Asp Phe Glu Thr Phe Tyr Glu Ile Trp Glu Lys Phe Asp 1780 1785 1790 Pro Asp Ala Thr Gln Phe Ile Glu Tyr Cys Lys Leu Ala Asp Phe Ala 1795 1800 1805 Asp Ala Leu Glu His Pro Leu Arg Val Pro Lys Pro Asn Thr Ile Glu 1810 1815 1820 Leu Ile Ala Met Asp Leu Pro Met Val Ser Gly Asp Arg Ile His Cys 1825 1830 1835 1840 Leu Asp Ile Leu Phe Ala Phe Thr Lys Ala Val Leu Gly Asp Ser Gly 1845 1850 1855 Glu Leu Asp Ile Leu Arg Gln Gln Met Glu Glu Arg Phe Val Ala Ser 1860 1865 1870 Asn Pro Ser Lys Val Ser Tyr Glu Ala Tyr His Thr Thr Leu Arg Arg 1875 1880 1885 Asn Glu Glu Glu Val Ser Ala Val Val Leu Gln Arg Ala Tyr Arg Gly 1890 1895 1900 His Leu Ala Arg Arg Gly Phe Ile Cys Arg Lys Met Ala Ser Asn Lys 1905 1910 1915 1920 Leu Glu Asn Gly Gly Thr His Arg Asp Lys Lys Glu Ser Thr Pro Ser 1925 1930 1935 Thr Ala Ser Leu Pro Ser Tyr Asp Ser Val Thr Lys Pro Asp Lys Glu 1940 1945 1950 Lys Gln Gln Arg Ala Glu Glu Gly Arg Arg Glu Arg Ala Lys Arg Gln 1955 1960 1965 Lys Glu Val Arg Glu Ser Lys Cys 1970 1975 150 amino acids amino acid single linear peptide 10 Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg Glu Arg Arg Ser Ser Tyr 1 5 10 15 Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser Arg Ser Ser Arg Ile Phe 20 25 30 Pro Ser Leu Arg Arg Ser Val Lys Arg Asn Ser Thr Val Asp Cys Asn 35 40 45 Gly Val Val Ser Leu Ile Gly Pro Gly Ser His Ile Gly Arg Leu Leu 50 55 60 Pro Glu Val Lys Ile Asp Lys Ala Ala Thr Asp Ser Ala Thr Thr Glu 65 70 75 80 Val Glu Ile Lys Lys Lys Gly Pro Gly Ser Leu Leu Val Ser Met Asp 85 90 95 Gln Leu Ala Ser Tyr Gly Arg Lys Asp Arg Ile Asn Ser Ile Met Ser 100 105 110 Val Val Thr Asn Thr Leu Val Glu Glu Leu Glu Glu Ser Gln Arg Lys 115 120 125 Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn Thr Phe Leu Ile Trp Glu 130 135 140 Cys His Pro Tyr Trp Ile 145 150 140 amino acids amino acid single linear peptide 11 Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg Glu Arg Arg Ser Ser Tyr 1 5 10 15 Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser Arg Ser Ser Arg Ile Phe 20 25 30 Pro Ser Leu Arg Arg Ser Val Lys Arg Asn Ser Thr Val Asp Cys Asn 35 40 45 Gly Val Val Ser Leu Ile Gly Pro Gly Ser His Ile Gly Arg Leu Leu 50 55 60 Pro Glu Ala Thr Thr Glu Val Glu Ile Lys Lys Lys Gly Pro Gly Ser 65 70 75 80 Leu Leu Val Ser Met Asp Gln Leu Ala Ser Tyr Gly Arg Lys Asp Arg 85 90 95 Ile Asn Ser Ile Met Ser Val Val Thr Asn Thr Leu Val Glu Glu Leu 100 105 110 Glu Glu Ser Gln Arg Lys Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn 115 120 125 Thr Phe Leu Ile Trp Glu Cys His Pro Tyr Trp Ile 130 135 140 138 amino acids amino acid single linear peptide 12 Asp Ser Leu Phe Ile Pro Ile Arg Ala Arg Glu Arg Arg Ser Ser Tyr 1 5 10 15 Ser Gly Tyr Ser Gly Tyr Ser Gln Cys Ser Arg Ser Ser Arg Ile Ser 20 25 30 Pro Ala Cys Ala Gln Arg Glu Ala Asn Ser Thr Val Asp Cys Asn Gly 35 40 45 Val Val Ser Leu Ile Gly Pro Gly Ser His Ile Gly Arg Leu Leu Leu 50 55 60 Arg Gln Arg Leu Arg Trp Lys Leu Arg Arg Lys Ala Leu Asp Ser Phe 65 70 75 80 Ser Phe Tyr Gly Pro Thr Arg Leu Leu Arg Thr Glu Gly Gln Asn Gln 85 90 95 Gln His Asn Glu Arg Gly His Lys His Ala Ser Glu Glu Leu Glu Glu 100 105 110 Ser Gln Arg Lys Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn Thr Phe 115 120 125 Leu Ile Trp Glu Cys His Pro Tyr Trp Ile 130 135 138 amino acids amino acid single linear peptide 13 Asp Ser Leu Phe Val Pro His Arg His Gly Glu Arg Arg Pro Ser Asn 1 5 10 15 Val Ser Gln Ala Ser Arg Ala Ser Arg Gly Ile Pro Thr Leu Pro Met 20 25 30 Asn Gly Lys Met His Ser Ala Val Asp Cys Asn Gly Val Val Ser Leu 35 40 45 Val Gly Gly Pro Ser Ala Leu Thr Ser Pro Val Gly Gln Leu Leu Pro 50 55 60 Glu Gly Thr Thr Thr Glu Thr Glu Ile Arg Lys Arg Arg Ser Ser Ser 65 70 75 80 Tyr His Val Ser Met Asp Leu Leu Glu Asp Pro Ser Arg Gln Arg Ala 85 90 95 Met Ser Ile Ala Ser Ile Leu Thr Asn Thr Met Glu Glu Leu Glu Glu 100 105 110 Ser Arg Gln Lys Cys Pro Pro Cys Trp Tyr Lys Phe Ala Asn Met Cys 115 120 125 Leu Ile Trp Asp Cys Cys Lys Pro Trp Leu 130 135 20 base pairs nucleic acid single linear DNA (genomic) 14 CAATCGTGGG CGCCCTAATC 20 25 base pairs nucleic acid single linear DNA (genomic) 15 TGCTTTCATG CACTGGAATC CCTCT 25 25 base pairs nucleic acid single linear DNA (genomic) 16 TGCTTTACTG CACTGGAATC CTTCG 25 12 amino acids amino acid single linear DNA (genomic) 17 Gly Arg Leu Leu Pro Glu Ala Thr Thr Glu Val Glu 1 5 10 22 amino acids amino acid single linear DNA (genomic) 18 Gly Arg Leu Leu Pro Glu Val Lys Ile Asp Lys Ala Ala Thr Asp Ser 1 5 10 15 Ala Thr Thr Glu Val Glu 20 24 amino acids amino acid single linear DNA (genomic) 19 Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys Pro Ala Thr Asp Asp 1 5 10 15 Asn Gly Thr Thr Thr Glu Thr Glu 20 13 amino acids amino acid single linear DNA (genomic) 20 Gly Gln Leu Leu Pro Glu Gly Thr Thr Thr Glu Thr Glu 1 5 10 24 amino acids amino acid single linear DNA (genomic) 21 Gly Gln Leu Leu Pro Glu Val Ile Ile Asp Lys Ala Thr Ser Asp Asp 1 5 10 15 Ser Gly Thr Thr Asn Gln Met Arg 20 13 amino acids amino acid single linear DNA (genomic) 22 Gly Gln Leu Leu Pro Glu Gly Thr Thr Asn Gln Ile His 1 5 10 13 amino acids amino acid single linear DNA (genomic) 23 Gly Gln Leu Leu Pro Glu Gly Thr Thr Thr Glu Thr Glu 1 5 10 7 amino acids amino acid single linear DNA (genomic) 24 Gly Thr Thr Thr Glu Thr Glu 1 5 7 amino acids amino acid single linear DNA (genomic) 25 Thr Thr Pro Ser Glu Glu Pro 1 5 21 base pairs nucleic acid single linear DNA (genomic) 26 ACACTCAGAG CAAGCAGATG G 21 21 base pairs nucleic acid single linear DNA (genomic) 27 TCCCTGGGTG CTCTTTGTCC A 21 20 base pairs nucleic acid single linear DNA (genomic) misc_difference replace(9, “”) /note= “This position is T or C.” misc_difference replace(12, “”) /note= “This position is G or A.” misc_difference replace(15, “”) /note= “This position is T or C.” 28 ACCAACTGYG TRTTYATGAC 20 20 base pairs nucleic acid single linear DNA (genomic) 29 CAGCAGCTAC AGTGGCTACA 20 20 base pairs nucleic acid single linear DNA (genomic) 30 AAAGAGGCCG AGTTCAAGGC 20 18 base pairs nucleic acid single linear DNA (genomic) 31 TGTCCTTCCG TCCGTAGG 18 21 base pairs nucleic acid single linear DNA (genomic) 32 TTCATGGGGA ACCTTCGAAA C 21 25 base pairs nucleic acid single linear DNA (genomic) 33 GAACGATGCA GATGGTGATG GCTAA 25 37 base pairs nucleic acid single linear DNA (genomic) 34 GAAGCTCGAG CCCGGGCAAG AGAAGATGGC AGCGCGG 37 20 base pairs nucleic acid single linear DNA (genomic) 35 CTCGGAGAGC CTACCCCATC 20 21 base pairs nucleic acid single linear DNA (genomic) 36 AGAAGGGGAA GATGGGGTAG G 21 21 base pairs nucleic acid single linear DNA (genomic) 37 ATTCTGTCCT TCCGTCCGTA G 21 21 base pairs nucleic acid single linear DNA (genomic) 38 ACACTCAGAG CAAGCAGATG G 21 21 base pairs nucleic acid single linear DNA (genomic) 39 TCCCTGGGTG CTCTTTGTCC A 21 19 base pairs nucleic acid single linear DNA (genomic) 40 GGTGGACTGC AACGGCGTA 19 21 base pairs nucleic acid single linear DNA (genomic) 41 ATTCTGTCCT TCCGTCCGTA G 21 30 base pairs nucleic acid single linear DNA (genomic) 42 GTGAAAATAG ATAAGGCAGC TACGGACAGC 30 6586 base pairs nucleic acid single linear DNA (genomic) 43 CCAAGATGGC GCCCACCGCA GTCCCGCCCG CCGCAGCCTC GGCGCCTCTG CAGTCCGGCC 60 GCGCCTCCCG GGCCCCGCGC TAGGGCCGCT GCCGCCTCGC CCGCCGCCGC CGCCGCCAGC 120 TGACCTGTCC CGGACACATA ACTAACGAAG CTGCTGCAGG ATGAGAAGAT GGCAGCGCGG 180 CTGCTCGCAC CACCAGGCCC TGATAGTTTC AAGCCTTTCA CCCCTGAGTC GCTGGCAAAC 240 ATCGAGAGGC GTATTGCCGA GAGCAAGCTC AAGAAACCAC CAAAGGCGGA TGGCAGCCAC 300 CGGGAGGACG ATGAAGACAG CAAGCCCAAG CCAAACAGTG ACCTGGAGGC TGGGAAGAGT 360 TTGCCTTTCA TCTACGGGGA CATCCCGCAA GGCCTGGTTG CGGTTCCCCT GGAGGACTTT 420 GACCCTTACT ATTTGACGCA GAAAACCTTT GTAGTATTAA ACAGAGGGAA AACTCTCTTC 480 AGATTTAGTG CCACACCTGC CTTGTACATT TTAAGCCCTT TTAACCTGAT AAGAAGAATA 540 GCTATTAAAA TTTTGATACA CTCAGTTTTC AGCATGATCA TCATGTGCAC CATCCTGACC 600 AACTGTGTGT TCATGACCTT TAGTAACCCT CCAGAATGGT CCAAGAATGT GGAGTACACA 660 TTCACAGGGA TTTACACATT TGAATCACTA GTGAAAATCA TCGCAAGAGG TTTCTGCATA 720 GACGGCTTCA CCTTCTTGCG AGACCCGTGG AACTGGTTAG ACTTCAGTGT CATCATGATG 780 GCATATGTGA CAGAGTTTGT GGACCTGGGC AATGTCTCAG CGCTGAGAAC ATTCAGGGTT 840 CTCCGAGCTT TGAAAACTAT CTCTGTAATT CCAGGCCTGA AGACAATCGT GGGCGCCCTA 900 ATCCAGTCCG TGAAGAAGCT GTCGGACGTG ATGATCCTGA CAGTGTTCTG CCTGAGTGTT 960 TTCGCCCTGA TTGGCCTGCA GCTCTTCATG GGGAACCTTC GAAACAAGTG TGTCGTGTGG 1020 CCCATAAACT TCAACGAGAG CTACCTGGAG AACGGCACCA GAGGCTTTGA CTGGGAGGAA 1080 TATATCAACA ATAAAACAAA CTTTTACATG GTTCCTGGCA TGCTAGAACC CTTGCTCTGC 1140 GGGAACAGTT CTGATGCTGG GCAATGCCCA GAGGGATTCC AGTGCATGAA AGCAGGAAGG 1200 AACCCCAACT ACGGTTACAC CAGCTTTGAC ACCTTCAGCT GGGCCTTCTT GGCATTATTC 1260 CGCCTTATGA CCCAGGACTA TTGGGAGAAC TTATACCAGC TGACCTTACG AGCCGCTGGG 1320 AAAACGTACA TGATCTTCTT TGTCTTGGTC ATCTTCGTGG GTTCTTTCTA TCTGGTGAAC 1380 TTGATCTTGG CTGTGGTGGC CATGGCTTAT GAGGAACAGA ACCAGGCAAC ACTGGAGGAG 1440 GCAGAGCAAA AAGAGGCCGA GTTCAAGGCA ATGCTGGAGC AACTCAAGAA GCAGCAGGAG 1500 GAGGCACAGG CTGCTGCAAT GGCCACCTCA GCGGGCACTG TCTCGGAAGA CGCCATTGAA 1560 GAAGAAGGGG AAGATGGGGT AGGCTCTCCG AGGAGCTCTT CTGAACTGTC TAAACTCAGT 1620 TCCAAGAGCG CGAAGGAGCG GCGGAACCGA CGGAAGAAGA GGAAGCAGAA GGAGCTCTCT 1680 GAAGGCGAGG AGAAAGGGGA CCCGGAGAAG GTGTTTAAGT CAGAGTCGGA AGACGGTATG 1740 AGAAGGAAGG CCTTCCGGCT GCCAGACAAC AGGATAGGGA GGAAGTTTTC CATCATGAAT 1800 CAGTCGCTGC TCAGCATTCC AGGCTCGCCC TTCCTCTCCC GACATAACAG CAAAAGCAGC 1860 ATCTTCAGCT TCCGGGGACC CGGTCGGTTC CGGGACCCCG GCTCTGAGAA TGAGTTCGCA 1920 GACGATGAAC ACAGCACCGT GGAGGAGAGC GAGGGCCGGC GTGACTCGCT CTTCATCCCG 1980 ATCCGCGCCC GCGAGCGCCG CAGCAGCTAC AGTGGCTACA GCGGCTACAG CCAGTGCAGC 2040 CGCTCGTCGC GTGAAAATAG ATAAGGCAGC TACGGACAGC GCATCTTCCC CAGCCTGCGG 2100 CGCAGCGTGA AGCGCAACAG CACGGTGGAC TGCAACGGCG TAGTGTCACT CATCGGGCCC 2160 GGCTCACACA TCGGGCGGCT CCTGCCTGAG GCAACGACTG AGGTGGAAAT TAAGAAGAAA 2220 GGCCCTGGAT CTCTTTTAGT TTCTATGGAC CAACTCGCCT CCTACGGACG GAAGGACAGA 2280 ATCAACAGCA TAATGAGCGT GGTCACAAAC ACGCTAGTGG AAGAGCTGGA AGAGTCTCAG 2340 AGAAAGTGCC CACCGTGCTG GTATAAGTTT GCCAACACTT TCCTCATCTG GGAGTGTCAC 2400 CCCTACTGGA TAAAACTGAA GGAGATCGTG AACTTAATCG TCATGGACCC TTTTGTAGAC 2460 TTAGCCATCA CCATCTGCAT CGTTCTGAAT ACGCTATTTA TGGCAATGGA GCACCATCCC 2520 ATGACACCAC AGTTCGAACA CGTCTTGGCC GTAGGAAATC TGGTGTTCAC CGGGATCTTC 2580 ACGGCGGAAA TGTTTCTGAA GCTCATAGCC ATGGACCCCT ACTATTATTT CCAAGAAGGC 2640 TGGAACATTT TTGACGGATT TATTGTCTCC CTCAGTTTAA TGGAGCTGAG TCTCGCAGAT 2700 GTGGAGGGGC TCTCAGTGCT GCGGTCTTTC CGACTGCTCC GAGTCTTCAA GCTGGCCAAG 2760 TCCTGGCCCA CCCTGAACAT GCTGATCAAG ATCATCGGGA ACTCCGTGGG TGCCCTGGGC 2820 AACCTGACCC TGGTGCTGGC CATCATCGTC TTCATCTTCG CCGTGGTGGG GATGCAGCTG 2880 TTTGGAAAGA GTTACAAGGA GTGCGTCTGT AAGATCAACC AGGAGTGCAA GCTCCCGCGC 2940 TGGCACATGA ACGACTTCTT CCACTCCTTC CTCATCGTCT TCCGAGTGCT GTGTGGGGAG 3000 TGGATCGAGA CCATGTGGGA CTGCATGGAG GTGGCCGGCC AGGCCATGTG CCTCATTGTC 3060 TTCATGATGG TTATGGTCAT TGGCAACCTG GTGGTGCTGA ATCTATTCCT GGCCTTGCTT 3120 CTGAGCTCCT TCAGCGCAGA CAACCTGGCG GCCACAGACG ACGACGGGGA AATGAACAAC 3180 CTGCAGATCT CAGTGATCCG GATCAAGAAG GGCGTGGCCT GGACCAAAGT GAAGGTGCAC 3240 GCCTTCATGC AGGCTCACTT CAAGCAGCGG GAGGCGGATG AAGTGAAACC CCTCGACGAG 3300 CTGTATGAGA AGAAGGCCAA CTGCATCGCC AACCACACGG GCGTGGATAT CCACCGGAAC 3360 GGCGACTTCC AGAAGAACGG GAACGGAACC ACCAGCGGCA TCGGCAGCAG CGTGGAGAAG 3420 TACATCATCG ACGAGGACCA CATGTCCTTC ATTAACAACC CAAACCTGAC CGTCCGGGTG 3480 CCCATTGCTG TGGGCGAGTC TGACTTCGAG AACCTCAACA CAGAGGATGT TAGCAGCGAA 3540 TCAGACCCTG AAGGCAGCAA AGATAAACTG GACGATACCA GCTCCTCAGA AGGAAGTACC 3600 ATCGACATCA AGCCTGAGGT GGAAGAAGTT CCCGTGGAGC AACCTGAGGA ATACTTGGAT 3660 CCGGACGCCT GCTTTACAGA GGGTTGCGTC CAGCGGTTCA AGTGCTGCCA GGTCAACATC 3720 GAGGAAGGAC TAGGCAAGTC GTGGTGGATC TTGCGGAAAA CCTGCTTCCT CATTGTGGAG 3780 CACAATTGGT TTGAGACCTT CATCATCTTC ATGATTCTGC TCAGCAGTGG CGCCCTGGCC 3840 TTTGAGGACA TCTACATTGA GCAGAGGAAG ACCATCCGCA CCATCCTGGA GTATGCGGAC 3900 AAGGTCTTCA CCTACATCTT CATCCTGGAG ATGTTGCTCA AGTGGACAGC CTACGGCTTC 3960 GTCAAGTTCT TCACCAATGC CTGGTGCTGG TTGGACTTCC TCATTGTGGC TGTCTCTTTA 4020 GTCAGCCTTA TAGCTAATGC CCTGGGCTAC TCGGAACTAG GTGCCATAAA GTCCCTTAGG 4080 ACCCTAAGAG CTTTGAGACC CTTAAGAGCC TTATCACGAT TTGAAGGGAT GAGGGTGGTG 4140 GTGAATGCCT TGGTGGGCGC CATCCCCTCC ATCATGAATG TGCTGCTGGT GTGTCTCATC 4200 TTCTGGCTGA TTTTCAGCAT CATGGGAGTT AACCTGTTTG CGGGGAAATA CCACTACTGC 4260 TTTAATGAGA CTTCTGAAAT CCGGTTCGAA ATCGATATTG TCAACAATAA AACGGACTGT 4320 GAGAAGCTCA TGGAGGGCAA CAGCACGGAG ATCCGATGGA AGAATGTCAA GATCAACTTT 4380 GACAATGTCG GAGCAGGGTA CCTGGCCCTT CTTCAAGTGG CAACCTTCAA AGGCTGGATG 4440 GACATCATGT ATGCGGCTGT AGATTCCCGA AAGCCAGACG AGCAGCCTGA CTACGAGGGC 4500 AACATCTACA TGTACATCTA CTTCGTCATC TTCATCATCT TCGGCTCCTT CTTCACCCTC 4560 AACCTGTTCA TCGGTGTCAT CATCGACAAC TTCAACCAGC AGAAGAAAAA GTTTGGAGGT 4620 CAGGACATCT TCATGACAGA GGAACAGAAG AAGTACTACA ATGCCATGAA AAAGCTGGGC 4680 TCCAAGAAGC CACAGAAGCC CATCCCCCGA CCCTTGAACA AAATCCAAGG GATTGTCTTT 4740 GATTTCGTCA CTCAACAAGC CTTTGACATT GTGATCATGA TGCTCATCTG CCTTAACATG 4800 GTGACAATGA TGGTGGAGAC AGACACTCAG AGCAAGCAGA TGGAGAACAT TCTTTACTGG 4860 ATTAATCTGG TCTTTGTCAT CTTCTTCACC TGCGAGTGTG TGCTCAAAAT GTTTGCCTTG 4920 AGACACTACT ATTTCACCAT TGGCTGGAAC ATCTTTGACT TTGTGGTGGT CATCCTCTCC 4980 ATTGTGGGAA TGTTCCTGGC TGATATCATT GAGAAGTACT TCGTCTCCCC AACCCTATTC 5040 CGAGTTATCC GATTGGCCCG TATTGGGCGC ATCTTGCGTC TGATCAAGGG CGCCAAAGGG 5100 ATCCGCACCC TGCTCTTTGC CTTAATGATG TCGCTGCCCG CCCTGTTCAA CATCGGCCTC 5160 CTGCTCTTCC TCGTCATGTT CATCTTCTCC ATTTTTGGCA TGTCCAACTT CGCATACGTG 5220 AAGCACGAGG CCGGCATTGA CGACATGTTC AACTTCGAGA CATTTGGCAA CAGCATGATC 5280 TGTTTGTTCC AGATCACAAC GTCTGCTGGC TGGGATGGCC TGCTGCTGCC AATCCTGAAC 5340 CGCCCCCCTG ACTGCAGCTT GGACAAAGAG CACCCAGGGA GTGGCTTCAA AGGGGACTGT 5400 GGGAACCCCT CGGTGGGCAT CTTCTTCTTT GTGAGCTACA TCATCATCTC CTTCCTGATT 5460 GTGGTGAACA TGTACATCGC CATCATCCTG GAGAACTTCA GCGTGGCCAC CGAGGAGAGC 5520 GCCGACCCTC TGAGTGAGGA TGACTTCGAG ACTTTCTATG AGATCTGGGA GAAGTTTGAC 5580 CCAGACGCCA CCCAGTTCAT CGAGTACTGT AAGCTGGCAG ACTTTGCCGA CGCCCTGGAG 5640 CACCCGCTCC GAGTACCCAA GCCCAACACC ATCGAGCTCA TCGCCATGGA CCTGCCCATG 5700 GTGAGCGGAG ATCGCATCCA CTGCTTGGAC ATCCTTTTCG CCTTCACCAA GCGAGTCCTG 5760 GGAGACAGTG GGGAGTTGGA CATCCTGCGG CAGCAGATGG AGGAGCGGTT CGTGGCATCC 5820 AATCCTTCCA AAGTGTCTTA CGAGCCTATC ACAACCACTC TGCGGCGCAA GCAGGAGGAG 5880 GTGTCTGCAG TGGTCCTGCA GCGTGCCTAC AGGGGACACT TGGCTAGGCG GGGCTTCATC 5940 TGCAGAAAGA TGGCCTCCAA CAAGCTGGAG AATGGAGGCA CACACAGAGA CAAGAAGGAG 6000 AGCACCCCGT CCACAGCCTC CCTCCCCTCT TACGACAGCG TCACAAAGCC AGACAAGGAG 6060 AAGCAGCAGC GTGCGGAGGA GGGCAGAAGG GAAAGAGCCA AGAGGCAAAA AGAGGTCAGG 6120 GAGTCCAAGT GCTAGAGGAG GGGAAAGGAA GCTTACCCCG GCTGAACACT GGCAAGTGAA 6180 AGCTTGTTTA CAAACTTCCG AATCTCACGG ATGCAGAGCA GCTGTGCAGA CGCTCGCTGT 6240 ACTGGAAGAC CTATACCAAA CATAGTCTGC TTACATGTGA CATGGTGGCA TCCTGAGCGG 6300 TGACTGCTGG GGACAAAGGA CCCTGCTCCC TGGACTCACA GATCTCCTAT CGCTTGGGCA 6360 GACGGTTACT GCATGTTCCA CACTTAGTCA ATGCAACTTA GGACTAAACT AACCAGGATA 6420 CAAAACCGAG GCGGCTGCCG GGACCAGCAG ATCACCGCTG CAGCCAAATG GATTTTATTT 6480 TTTCATTTTG TTGATTCTCA GAAGCAGAAA GCATCACTTT AAAAGTTTGT TTGTTCATNC 6540 AAACAATATT TGAATTCTTA CATTAGTTAA GCTAAGCANC AAAAAG 6586 

What is claimed is:
 1. An isolated tetrodotoxin-sensitive sodium channel α-subunit protein encoded by a DNA comprising SEQ ID NO:1.
 2. An isolated tetrodotoxin-sensitive sodium channel α-subunit protein comprising the amino acid sequence set forth in SEQ ID NO:3.
 3. An assay for inhibitors of the tetrodotoxin-sensitive sodium channel α-subunit protein of claim 2, comprising contacting a compound suspected of being said inhibitor with said sodium channel protein and measuring the activity of said sodium channel α-subunit protein.
 4. An isolated tetrodotoxin-sensitive sodium channel α-subunit protein encoded by a DNA comprising SEQ ID NO:2.
 5. The protein of claim 4 comprising the amino acid sequence of SEQ ID NO:4.
 6. An assay for inhibitors of the tetrodotoxin-sensitive sodium channel α-subunit protein of claim 5, comprising contacting a compound suspected of being an inhibitor of said sodium channel α-subunit protein and measuring the activity of said sodium channel α-subunit protein. 